Structural and Functional Characterization of The Surface Layer Protein of Lactobacillus Brevis ATCC 8287

Structural and functional characterization of the surface layer protein of Lactobacillus brevis ATCC 8287
Structural and functional characterization

of the surface layer protein of Lactobacillus brevis
ATCC 8287
Ulla Hynönen
Department of Basic Veterinary Sciences

University of Helsinki
Helsinki 2009
Ulla Hynönen
University of Helsinki
Helsinki
Structural and functional characterization

of the surface layer protein of Lactobacillus brevis
ATCC 8287
Ulla Hynönen
ACADEMIC DISSERTATION
To be presented, with the permission of the Faculty of Veterinary Medicine,

for public examination in the Walter Auditorium, Agnes Sjöberginkatu 2, Helsinki,
on December 4th 2009, at 12 o’clock noon.
Helsinki 2009
Supervisors Professor Timo K. Korhonen
General Microbiology
Department of Biological and Environmental Sciences
University of Helsinki, Finland
Professor Airi Palva

Microbiology and Epidemiology
Reviewers Professor Per Saris

Microbiology
Department of Applied Chemistry and Microbiology
Professor Neil F. Fairweather

Division of Cell and Molecular Biology
Centre for Molecular Microbiology and Infection
Imperial College London, UK
Opponent Professor Atte von Wright

Institute of Applied Biotechnology
Department of Biosciences
University of Kuopio, Finland
Layout: Tinde Päivärinta
ISBN 978-952-92-6380-6 (paperback)

ISBN 978-952-10-5852-3 (PDF)
http://ethesis.helsinki.fi
Yliopistopaino
Helsinki 2009
CONTENTS
SUMMARY
LIST OF ORIGINAL PUBLICATIONS
ABBREVIATIONS
1. REVIEW OF THE LITERATURE ........................................................................ 1
1.1 Introduction ........................................................................................................ 1
1.1.1 Structure of the Gram-positive cell wall ................................................... 1
1.1.2 Surface layer proteins ................................................................................ 3
1.1.2.1 Occurrence, general features and study methods .......................... 3
1.1.2.2 Expression of S-layer protein genes ............................................. 5
1.1.2.3 Cell wall binding and self-assembly regions ................................ 6
1.1.2.4 Functions ....................................................................................... 8
1.1.2.5 Applications .................................................................................. 9
1.2 Lactobacilli and their S-layer proteins ............................................................. 10
1.2.1 Occurrence and general properties of Lactobacillus S-layer proteins .... 11
1.2.2 Expression of Lactobacillus S-layer protein genes ................................. 13
1.2.3 Cell wall binding and self-assembly regions of Lactobacillus S-layer
proteins .................................................................................................... 15
1.2.4 Functions of Lactobacillus S-layer proteins ............................................ 17
1.2.5 Applications of Lactobacillus S-layer proteins ....................................... 19
1.2.6 Tools to study S-layer structure and function: cysteine scanning
mutagenesis and bacterial surface display ............................................. 20
1.2.6.1 Cysteine scanning mutagenesis and sulfhydryl
chemistry ................................................................................................. 20
1.2.6.2 Flagellar diplay ........................................................................... 22
1.2.7 Non-S-layer adhesive surface proteins of lactobacilli ............................. 24
2. AIMS OF THE STUDY ......................................................................................... 29
3. MATERIALS AND METHODS ........................................................................... 30
4. RESULTS AND DISCUSSION ............................................................................. 32
4.1. Structure and function of SlpA ........................................................................ 32
4.1.1 Adhesive functions (I) ............................................................................. 42
4.1.2 Cell wall binding (II) ............................................................................... 35
4.1.3 Self-assembly (II) .................................................................................... 36
4.1.4 Locations of individual residues (III) ...................................................... 37
4.2. Activities of slpA promoters (IV) ..................................................................... 38
5. CONCLUSIONS .................................................................................................... 40
6. ACKNOWLEDGEMENTS .................................................................................. 42
7. REFERENCES ...................................................................................................... 43
SUMMARY
Bacterial surface (S) layers are proteinaceous arrays found on the surface of hundreds
of bacterial species, including several species of lactobacilli. They are composed of
numerous identical, non-covalently bound subunits, which completely cover the cell
surface forming a symmetric, porous, lattice-like structure. Several functions for S-layers
have been found, but no common one probably exists. S-layer proteins have a wide
application potential in nanobiotechnology as well as in health-related applications such
as vaccine design.
In this work, the structure and function of the S-layer protein SlpA of Lactobacillus
brevis ATCC 8287 and the expression of the slpA gene were studied. SlpA was identified
as a two-domain protein, in which the N-terminal domain is responsible for binding to
the cell wall and the C-terminal domain for forming the regular polymer. The domain
organization is thus reversed compared with other hitherto characterized Lactobacillus
S-layer proteins. Conserved carbohydrate binding motifs were identified in the N-terminal,
positively charged amino acid sequences of SlpA and five other Lactobacillus brevis
S-layer proteins. The component in the cell wall interacting with SlpA was shown to be
something other than teichoic or lipoteichoic acid, in contrast to the cell wall receptors of
S-layer proteins previously characterized in lactobacilli. The structure of the C-terminal
self-assembly domain was studied in more detail using cysteine scanning mutagenesis
and targeted chemical modification. Importantly for the potential future applications of
SlpA as a display vehicle of foreign peptides, four amino acid segments with high surface
accessibility in the assembled form of SlpA were detected. The 46 mutated residues
could be grouped according to their location in the lattice: in the protein interior, on the
inner surface of the lattice, on the outer surface of the lattice and on the subunit interface
or the pore region of the lattice.
L. brevis ATCC 8287 very efficiently adheres to cultured human epithelial cells
representing the human gut, bladder and blood vessels, while the removal of the S-layer
abolishes the binding. This binding was shown to be mediated by SlpA by using flagellum
display. Hybrid flagella carrying fragments from the N-terminal part of SlpA bound to
epithelial cells and to fibronectin, while flagella carrying the C-terminal part were unable
to bind. The smallest fragment conferring binding to Int 407 cells comprised amino acids
66-215 in mature SlpA.
The gene encoding SlpA is preceded by two promoters. By separating them on
reporter plasmids, both of the promoters were shown to be used in L. brevis in all
growth phases. More upstream region was needed for the full activity of the upstream
promoter than for the downstream promoter. The promoter activities seen at the reporter
enzyme level were also seen at the mRNA level, suggesting transcriptional rather than
translational regulation of slpA. Three potential regulatory motifs were identified in
the upstream region of slpA. Both promoters retained their activities under selected
conditions mimicking the intestinal environment in vitro.
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following articles, referred to in the text by their Roman
numerals. These original articles are reprinted with the kind permission of their copyright
holders.
I Hynönen, U., B. Westerlund-Wikström, A. Palva, and T. K. Korhonen.

2002. Identification by flagellum display of an epithelial cell- and fibronectin-
binding function in the SlpA surface protein of Lactobacillus brevis. J. Bacteriol.
184:3360-3367.
II Åvall-Jääskeläinen, S., U. Hynönen, N. Ilk, D. Pum, U. B. Sleytr, and A.

Palva. 2008. Identification and characterization of domains responsible for self-
assembly and cell wall binding of the surface layer protein of Lactobacillus
brevis ATCC 8287. BMC Microbiol. 8:165.
III Vilen, H., U. Hynönen, H. Badelt-Lichtblau, N. Ilk, P. Jääskeläinen, M.

Torkkeli, and A. Palva. 2009. Surface location of individual residues of SlpA
provides insight into Lactobacillus brevis S-layer. J. Bacteriol. 191:3339-3349.
IV Hynönen, U., S. Åvall-Jääskeläinen, and A. Palva. 2009. Characterization

and separate activities of the two promoters of the Lactobacillus brevis S-layer
protein gene. Submitted manuscript.
ABBREVIATIONS
Aa amino acid
AAD antibiotic-associated diarrhoea
AFM atomic force microscopy
Alexa AlexaFluor488 C5-maleimide
APC antigen presenting cell
ATCC American Type Culture Collection
BMP bacterial magnetic particle
bp base pair
BSA bovine serum albumin
CRE catabolite response element
Cryo-EM cryoelectron microscopy
CSM cysteine scanning mutagenesis
C-terminus carboxyterminus
CW, CWF cell wall fragment
Da dalton
DMSO dimethyl sulfoxide
DNA deoxyribonucleic acid
DNAase deoxyribonuclease
DTT dithiothreitol
EM electron microscopy
FTIR Fourier transform infrared
GFP Green Fluorescent Protein
GnHCl, GHCl guanidine hydrochloride
GRAS generally recognized as safe
Hepes 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulphonic acid
IEM immunoelectron microscopy
IPTG isopropyl-β-D-thiogalactopyranoside
kb kilobase pair
LTA lipoteichoic acid
Mono monomeric
mPEG-maleimide methyl-capped polyethylene glycol maleimide
mRNA messenger ribonucleic acid
MW molecular weight
NEM N-ethyl maleimide
NEXAFS near-edge X-ray absorption fine structure
NMR nuclear magnetic resonance
N-terminus aminoterminus
OMP outer membrane protein
ORF open reading frame
P promoter
PBS phosphate-buffered saline
PCR polymerase chain reaction
PE photoemission
PEG polyethylene glycol
pI isolelectric point
PIPES 1,4-piperazinediethanesulphonic acid
RBS ribosome binding sequence
RNA ribonucleic acid
RNAase ribonuclease
rRNA ribosomal ribonucleic acid
rSlpA recombinant SlpA
SAXS small angle X-ray scattering
SCWP secondary cell wall polymer
SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis
SELDI-TOF surface-enhanced laser desorption/ionization-time of flight
SFM scanning force microscopy
S-layer surface layer
SLH S-layer homology
SS signal sequence
t transcription terminator sequence
TCA trichloroacetic acid
TEM transmission electron microscopy
TMM(PEG)12 trimethyl maleimide polyethylene glycol,
(methyl-PEG12)3-PEG4-maleimide
TRAC transcript analysis with aid of affinity capture
TREC topography and recognition imaging
UTR untranslated region
vol volume
wt weight, wild type
Å ångström, 0.1 nm
Review of the Literature
1. REVIEW OF THE LITERATURE
1.1 Introduction
1.1.1 Structure of the Gram-positive cell wall

The bacterial cell envelope consists of the cytoplasmic membrane and the overlying cell
wall. The cell walls of Gram-negative and Gram-positive bacteria differ fundamentally
in several respects: while the cell walls of Gram-negative bacteria are composed of
a thin peptidoglycan layer covered by the outer membrane, the Gram-positive cell
wall has no outer membrane and is characterized by a very thick peptidoglycan
layer and abundant Gram-positive specific cell wall carbohydrates. Peptidoglycan is
composed of glycan strands of variable length with alternating N-acetyl-muramic acid
and N-acetyl-glucosamine molecules, which are interconnected by short peptides.
According to the conventional model, this mesh-like structure lies horizontally to the
cell surface, and in Gram-positive cell walls multiple layers are present, interconnected
also in the vertical orientation. This mechanically very strong three-dimensional
network, the basic function of which is to provide protection and maintain the shape
of the cell, is decorated by other cell wall constituents, including proteins and teichoic
and lipoteichoic acids, lipoglycans, teichuronic acids and other acidic or neutral
polysaccharides (Delcour et al., 1999; Navarre & Schneewind, 1999; Ton-That et
al., 2004; Holst & Müller-Loennies, 2007). In addition, capsular polysaccharides,
forming a thick outermost
polysaccharide layer, as well as
exopolysaccharides are present
S S S in many Gram-positive species
(Holst & Müller-Loennies,
2007). Polyglutamate capsules
LTA
are also sometimes present
WPS TA
protein
Figure 1. A schematic picture
showing the typical constituents
of the cell envelope of an
S-layered Gram-positive
PG bacterium. The figure is not
drawn to scale.
CM, cytoplasmic membrane;
lipoprotein
PG, peptidoglycan; S,
S-layer protein; WPS,
wall polysaccharide; LTA,
lipoteichoic acid; TA,
MP
teichoic acid; MP, membrane
CM
protein. Teichuronic acids,
lipoglycans and capsular or
exopolysaccharides are not
depicted.
1
(Candela & Fouet, 2006). Teichoic acids, teichuronic acids and polysaccharides are
collectively referred to as secondary cell wall polymers (SCWPs), and teichoic acids
and teichuronic acids have been called “classical” SCWPs (Schäffer & Messner, 2005).
Often also the membrane-linked components, lipoglycans, present mostly in high-
GC% Gram-positive bacteria such as bifidobacteria (Fischer, 1994), and lipoteichoic
acids have been included in SCWPs (Sara, 2001; Desvaux et al., 2006; Dramsi et al.,
2008). A schematic presentation of the typical Gram-positive cell wall is shown in
Fig.1.
The proteins anchored to the Gram-positive cell wall have been reviewed
elsewhere (Desvaux et al., 2006; Scott & Barnett, 2006), and the outermost protein
layer frequently present in the cell envelope, the surface (S) layer, will be discussed
in detail in Sections 1.1.2 and 1.2. The most often occurring teichoic acids are
polyol (usually glycerol or ribitol) phosphate or glycosylpolyol phosphate polymers,
typically substituted by glucose and/or esterified by alanine and covalently attached
to the muramic acid molecules of peptidoglycan through a glycosidic linkage unit.
Lipoteichoic acids essentially differ from teichoic acids only in that they are attached
to the cytoplasmic membrane via a glycolipid anchor, which is a diacylglycerol
molecule bound to a di- or trisaccharide. Due to the abundance of phosphate groups,
both types of molecules are highly negatively charged, the charge being regulated
by the level of D-alanylation (Delcour et al., 1999; Navarre & Schneewind, 1999;
Naumova et al., 2001; Holst & Müller-Loennies, 2007). Owing to the covalent linkage
to peptidoglycan, teichoic and teichuronic acids are sometimes collectively called
wall teichoic acids. Teichuronic acids are, however, completely different in structure,
as they are composed of sugar monomers directly linked by glycosidic bonds and
usually no linkage unit is present (Araki & Ito, 1989; Delcour et al., 1999). They
are devoid of phosphate groups; instead, in the teichuronic acids studied thus far, the
negative charges are provided by the carboxyl groups of uronic, usually glucuronic
or mannosamine uronic, acid residues. Teichuronic acids have been described in
Bacillus, Micrococcus and Streptomyces species (Hase & Matsushima, 1972; Ward,
1981; Shashkov et al., 2002), and according to some classifications (Sara, 2001), also
in Geobacillus (Schäffer et al., 1999); in B. subtilis, they replace teichoic acids under
phosphate-deprivation conditions (Lang et al., 1982). They are, however, likely to
occur also in lactobacilli (Delcour et al., 1999), although until now they have not been
described in lactic acid bacteria.
The “non-classical SCWPs” or “wall polysaccharides” are distinguished from
capsular polysaccharides, which form an outermost, thick, hydrated shell covalently
or non-covalently bound to the cell surface, and from exopolysaccharides (slimes),
which are released to the medium (Delcour et al., 1999; Holst & Müller-Loennies,
2007). Generally, they include glycosyl phosphate polymers (according to Araki and
Ito, 1989, however, classified as teichoic acids) and anionic or neutral sugar polymers
and are covalently attached to peptidoglycan. The sugar polymers are composed of
repeated sugar units, where the negative charge very often present arises from acidic
substituents such as sulphate or glycerol-phosphate groups or organic acids (Hancock
& Poxton, 1988; Schäffer & Messner, 2005). The “non-classical” SCWPs of some
members of the family Bacillaceae having S-layers have been studied in detail
2
(Schäffer & Messner, 2005). The polysaccharides characterized were acidic or neutral
heteropolysaccharides composed of 2-15 repeating units with 2-5 sugars in each unit.
Six different monosaccharide constituents and both linear and branched chains were
detected, and the polysaccharides could be classified into three groups according to
the sugar backbone structures. The non-carbohydrate modifications decorating the
sugar backbone were pyruvate, phosphate or acetate.
1.1.2 Surface layer proteins
1.1.2.1 Occurrence, general features and study methods

Surface (S) layers are cell envelope structures ubiquitously found in Gram-positive
and Gram-negative bacterial species as well as in Archaea (Sara & Sleytr, 2000).
They form the outermost proteinaceous layer on the cell and are sometimes covered
only by capsules (Fouet et al., 1999). S-layers are composed of numerous identical
(glyco)protein subunits, 40-200 kDa in molecular weight, which completely cover the
cell surface, forming a crystalline, two-dimensional, regular and porous array with
oblique (p1, p2), square (p4) or hexagonal (p3, p6) symmetry. The subunits of bacterial
S-layers are held together and attached to the underlying cell surface by non-covalent
interactions, and they have an intrinsic ability to spontaneously form regular layers
either in solution or on a solid support after the removal of the disintegrating agent
(Sara & Sleytr, 2000). The recrystallization of Bacillus sphaericus S-layer protein
subunits on hydrophobic silicon surfaces has been studied in real time by atomic force
microscopy; the subunits are initially randomly adsorbed to the surface, assembled
into small crystalline patches and the number of the patches increases until finally a
monolayer is formed (Györvary et al., 2003). The reassembly of S-layer proteins in
solution (Teixeira et al., 2009) and on lipid membranes and polyelectrolyte layers has
also been studied in detail by biophysical methods (Weygand et al., 1999; Weygand
et al., 2000; Weygand et al., 2002; Delcea et al., 2008). However, the incorporation
of subunits into the growing S-layer on bacterial cells is largely unexplored. S-layer
proteins may be modified by phosphorylation or glycosylation (Sara & Sleytr, 2000).
Glycosylated S-layer proteins are very common among Archaea, but they are also found
in Gram-positive bacteria and have recently been detected in some Gram-negative
species. The bacterial S-layer glycan chains characterized to date are O-glycosidically
linked, linear or branched homo- or heterosaccharides of 50-150 glycoses, organized
into 15-50 repeating units. The general 1-10 % (wt/wt) degree of glycosylation of
bacterial S-layer glycoproteins may be dependent on growth conditions (Messner et
al., 2008).
The primary structures of bacterial S-layer proteins are similar in that they are
generally rich in acidic, hydrophobic and hydroxyl-containing amino acids, and
cysteines are very rarely found. The predicted pI values are usually in a weakly acidic
range. Sequence similarity between S-layer protein genes, if any, is typically found
only between the S-layer protein genes of closely related species (Boot & Pouwels,
1996; Sara & Sleytr, 2000). An exception are the so-called SLH (S-layer homology)
motifs (Lupas et al., 1994), which are, however, not present in all S-layer proteins
and are found in other proteins as well (see Section 1.1.2.3). Since a universal
3
“signature” for identifying a protein as an S-layer protein is lacking, the unambiguous

identification of surface-expressed S-layers still relies on transmission electron
microscopy (TEM) of whole bacterial cells or cell wall fragments. Several electron
microscopic techniques have been used, of which freeze-etching and freeze-drying
in combination with heavy metal shadowing are the most feasible for this purpose
(Schuster et al., 2006); thin sectioning (Jakava-Viljanen et al., 2002; Ventura et al.,
2002) and immunogold-labelling (Vidgren et al., 1992) have been used as well.
For further structural analysis of S-layer lattices or modified S-layer lattices, other
microscopical and biophysical methods have been applied, including cryo-electron
microscopy (Lembcke et al., 1993) and cryo-electron tomography (Trachtenberg
et al., 2000), cryoelectron microscopy of lipid monolayer crystals (Norville et al.,
2007), scanning force microscopy (SFM) (also known as atomic force microscopy
(AFM) (Müller et al., 1996; 1999; Scheuring et al., 2002; Györvary et al., 2003;
Schär-Zammaretti & Ubbink, 2003; Toca-Herrera et al., 2004; Martin-Molina et al.,
2006; Anselmetti et al., 2007; Verbelen et al., 2007; Dupres et al., 2009; Tang et al.,
2009) or its application TREC (topography and recognition imaging) (Tang et al.,
2008), electron microscopy of non-stained S-layer samples combined with electron
holography (Simon et al., 2004), photoemission (PE) and near-edge X-ray absorption
fine structure (NEXAFS) spectroscopy (Vyalikh et al., 2005; Kade et al., 2007) and
small-angle X-ray spectroscopy (SAXS) of S-layered bacterial cells or self-assembly
products (Aichmayer et al., 2006; P. Jääskeläinen, personal communication).
Secondary structures of S-layer proteins are difficult to predict because the
prediction algorithms are based on the available structures of very dissimilar types of
proteins. Circular dichroism (CD) measurements have been performed mainly for the
S-layer proteins of Bacillus species (Sara & Sleytr, 2000; Rüntzler et al., 2004). These
studies have revealed an α-helix content of approximately 20%, a β-sheet content
of 40%, and 5-45% aperiodic folding and β-turns in these proteins. The α-helices
were mostly predicted to reside in the N-terminal parts of the proteins (Sara & Sleytr,
2000). Elucidation of the tertiary structure of S-layer proteins has been hindered by
their molecular weights not being in the suitable range (<40 kDa) for nuclear magnetic
resonance (NMR) studies, and by their low solubility; more specifically, their tendency
to form two-dimensional lattices rather than three-dimensional crystals in solution.
Therefore, only two structures of bacterial S-layer protein fragments obtained by
X-ray crystallization have thus far been available (Pavkov et al., 2008; Fagan et al.,
2009). Therefore, physical methods, such as fluorescence spectroscopy (Rüntzler et
al., 2004), SAXS (Pavkov et al., 2008; Fagan et al., 2009), combined genetic and
biochemical approaches, such as cysteine scanning mutagenesis and chemical cross-
linking (Howorka et al., 2000; Kinns & Howorka, 2008), structure prediction by
the mean force method (Horejs et al., 2008) and electron microscopy (Norville et
al., 2007) have been applied to gain insight into the three-dimensional structures of
S-layer proteins. By calculating projection maps from electron micrographs of lipid
monolayer crystals, structural information down to a resolution of 7 Å has been
obtained (Norville et al., 2007).
4
1.1.2.2 Expression of S-layer protein genes

Many bacterial species have more than one S-layer gene, but not all of them are
necessarily expressed at the same time: both silent genes, antigenic variation based on
S-layer gene expression (reviewed by Boot & Pouwels, 1996; Sara & Sleytr, 2000),
alternative expression of S-layer protein genes in or ex vivo (reviewed by Fouet, 2009),
sequential expression during growth (Mignot et al., 2004) and, rarely, superimposed
S-layers (Cerquetti et al., 2000) or S-layers composed of two different S-layer proteins
(Rothfuss et al., 2006; Fagan et al., 2009; Goh et al., 2009) have been described.
As S-layer proteins typically account for 10-25% of total cellular protein (Boot &
Pouwels, 1996; Smit, 2008), the expression of S-layer protein genes and the secretion
of the proteins must be very efficient. With the exception of the S-layer proteins of
Caulobacter crescentus (Awram & Smit, 1998), Serratia marcescens (Kawai et al.,
1998) and Campylobacter fetus (Thompson et al., 1998), which are secreted through
the ATP-dependent type I machinery, and the S-layer protein of Campylobacter rectus,
which does not have an N-terminal signal peptide either (Wang et al., 1998), bacterial
S-layer proteins are secreted by the Sec-dependent, general secretory pathway (Sara
& Sleytr, 2000). The very efficient expression of S-layer protein genes is contributed
by their efficient promoters, a biased codon usage typical of efficiently transcribed
genes and by the long half-lives of S-layer gene transcripts, which, in some cases, may
be due to their long untranslated leader sequences (Boot & Pouwels, 1996; Boot et
al., 1996b). Furthermore, many S-layer protein genes are preceded by more than one
promoter, which may not only increase the transcription efficiency but also offers a
way to regulate the S-layer gene expression in response to, for instance, growth stage
(Adachi et al., 1989) or environmental conditions (Novotny et al., 2008). Chromosomal
rearrangements cause variation in S-layer gene expression in Campylobacter fetus
(Dworkin & Blaser, 1996; 1997), Geobacillus stearothermophilus (Egelseer et al.,
2001; Scholz et al., 2001) and lactobacilli (see Section 1.2.2). However, excluding
the thoroughly studied regulation of the S-layer protein genes of Bacillus anthracis
(reviewed by Mignot et al., 2004; Fouet, 2009), the modulation of bacterial S-layer
gene expression by soluble factors is poorly known. Carbon source regulates the
S-layer protein production in Corynebacterium strains (Soual-Hoebeke et al., 1999),
and molecular investigations have revealed the presence of a transcriptional activator
of the S-layer protein gene in this species (Soual-Hoebeke et al., 1999; Hansmeier et
al., 2006) as well as in Aeromonas salmonicida (Noonan & Trust, 1995). In Thermus
thermophilus, in addition to transcriptional regulation, translational autoregulation of
S-layer protein gene expression has been suggested, as the C-terminal fragment of
the S-layer protein SlpA specifically binds to the 5’ UTR of the slpA mRNA in vitro
(Fernandez-Herrero et al., 1997). Later, the 5’ UTR of the T. thermophilus S-layer
protein gene has been shown to be responsible for the growth phase-dependent
repression of the S-layer protein (Castan et al., 2001). The temperature-regulation
of sgsE in Geobacillus stearothermophilus NRS 2004/3a has been suggested to
occur at the transcriptional level (Novotny et al., 2004; Novotny et al., 2008). In
Clostridium difficile, the exposure to high osmolarity or antibiotics increases S-layer
gene expression, but the mechanism is not known (Deneve et al., 2008).
5
1.1.2.3 Cell wall binding and self-assembly regions

According to present knowledge, bacterial S-layer proteins in general have two structural
and functional regions: a region involved in the attachment of the S-layer subunit to
the cell envelope and a region involved in S-layer assembly. The S-layer proteins of
Gram-negative bacteria bind to the O-polysaccharide part of lipopolysaccharides of
the outer membrane (Griffiths & Lynch, 1990; Kokka et al., 1990; Walker et al., 1994;
Ford et al., 2007), and the S-layer protein of the Gram-positive Corynebacterium
glutamicum, which has an unusual mycolic acid-containing cell wall, binds to the
hydrophobic layer above the cytoplasmic membrane (Chami et al., 1997; Bayan et
al., 2003). The Hpi protein of the Gram-positive Deinococcus radiodurans is also in
contact with the underlying lipid-rich layer of the cell wall, but the contributions of
Hpi and the additional S-layer protein SlpA to cell wall anchoring of the S-layer are
not clear (Rothfuss et al., 2006). The known interactions between the S-layer protein
and the cell wall in Gram-positive bacteria are summarized in Table 1. In many
Gram-positive bacilli (Mesnage et al., 2000) and in the ancient thermophile Thermus
thermophilus (Olabarria et al., 1996; Cava et al., 2004), SLH motifs (Lupas et al.,
1994), 55-60 amino acids long and often located in the N-terminal part of the S-layer
protein, are responsible for the attachment of the subunit proteins to the cell wall. SLH
motifs are not restricted to S-layer proteins, but are found in hundreds of bacterial
(Gram-positive and -negative), archaeal, eukaryotic and even viral proteins (http://
pfam.sanger.ac.uk//family/slh, cited Aug 31, 2009). In most of the studied Bacillus,
Lysinibacillus, Geobacillus and Thermus species, the binding of the SLH motifs of the
S-layer protein has been shown to occur through a pyruvate-containing polysaccharide
receptor in the cell wall, while in Clostridium thermocellum F1 the binding of SLH
motifs to peptidoglycan has been demonstrated. In S-layers of Gram-positive bacteria
not having SLH motifs, the attachment to the cell wall has been proposed to be
mediated by an interaction between basic amino acids in the cell wall binding region
of the S-layer protein and negatively charged cell wall carbohydrates. For example,
the cell wall receptors of such S-layers in Geobacillus species characterized so far
contain mannuronic acid, and teichoic and lipoteichoic acids have been shown to
be the cell wall receptors of the S-layer proteins of Lactobacillus acidophilus and
L. crispatus (see Section 1.2.3). However, some cell wall polysaccharides of Gram-
positive bacteria proposed to be involved in S-layer binding have a net neutral charge
(Steindl et al., 2002; Schäffer & Messner, 2005). In any case, most interactions
characterized thus far between S-layer proteins and underlying cell wall polymers
can be considered lectin-like and a degree of specificity is recognized (Sara & Sleytr,
2000). At present, structures of secondary cell wall polymers are also available for
Gram-positive bacteria with structurally and/or genetically uncharacterized S-layer
proteins (Schäffer et al., 2000; Schäffer & Messner, 2005; Steindl et al., 2005).
Among Gram-positive bacteria, the self-assembly regions of S-layer proteins
have thus far been investigated in the S-layers of lactobacilli (see Section 1.2.3)
and in the S-layers of Bacillus anthracis, Lysinibacillus sphaericus and Geobacillus
stearothermophilus. These studies mainly rely on electron microscopy of recombinant
S-layer protein fragments, and the self-assembly region has been shown to be located
centrally or at the C- or N-terminus. The experimentally verified self-assembly regions
6
Table 1. Interactions of S-layer proteins of Gram-positive bacteria with the cell wall.
Strain S-layer Interaction Cell wall Reference
protein site in S-layer receptor
protein
Bacillus anthracis Sap, N-terminal, Pyruvic acid- Mesnage et al.,
EA1 three SLH containing 1999a; 2000
motifs polysaccharide
Lysinibacillus sphaericus SbpA N-terminal, Pyruvic acid- Ilk et al., 1999;
CCM 2177 three SLH containing Huber et al., 2005
motifs, one polysaccharide
SLH-like motif
Lysinibacillus sphaericus SlpC N-terminal, Polysaccharide Li et al., 2009
C3-41 three SLH
motifs, third
essential
Bacillus thuringiensis ssp. SlpA N-terminal, Pyruvic acid- Mesnage et al., 2001
galleriae NRRL 4045 three SLH containing
motifs polysaccharide
Geobacillus SbsB N-terminal, Pyruvic acid- Ries et al., 1997;
stearothermophilus PV72/ three SLH containing Sara et al., 1998;
p2 motifs polysaccharide Mader et al., 2004;
Rüntzler et al., 2004
Thermus thermophilus SlpAOne N-terminal Pyruvic acid- Olabarria et al.,
HB8 SLH motif containing 1996; Cava et al.,
polysaccharide 2004
Thermoanaerobacterium S-layer N-terminal, at Pyruvic acid- Brechtel & Bahl,
thermosulfurigenes EM1 protein least one SLH containing 1999; May et al.,
motif polysaccharide 2006
Clostridium thermocellum SlpA N-terminal, Not determined Lemaire et al., 1998
NCIMB 10682 three SLH
motifs
Clostridium thermocellum Slp1, C-terminal, Peptidoglycan Zhao et al., 2006
F1 Slp2 three SLH
motifs
Geobacillus SbsC N-terminal Mannuronic Egelseer et al., 1998;
stearothermophilus ATCC acid-containing Schäffer et al., 1999;
12980 polysaccharide Ferner-Ortner et al.,
2007
Geobacillus SbsD N-terminal Mannuronic Egelseer et al., 2001;
stearothermophilus ATCC (postulated) acid-containing Schäffer et al., 1999
12980/G+ polysaccharide
Geobacillus SbsA N-terminal Mannuronic Egelseer et al., 1998;
stearothermophilus PV72/ acid-containing Schäffer et al., 1999
p6 polysaccharide
Geobacillus SgsE N-terminal Mannuronic Schäffer et al., 1999;
stearothermophilus NRS (based on acid-containing Schäffer et al., 2002;
2004/3a sequence polysaccharide Schäffer et al., 2007
similarity)
Aneurinibacillus SatB Not known Neutral Steindl et al., 2002;
thermoaerophilus DSM polysaccharide Schäffer & Messner,
10155 2005
Corynebacterium PS2 C-terminal, Mycomembrane Chami et al., 1997
glutamicum hydrophobic containing
mycolic acids
(suggested)
Lactobacillus acidophilus SA C-terminal Teichoic acids Smit & Pouwels,
ATCC 4356 2002
Lactobacillus crispatus CbsA C-terminal Teichoic acids Antikainen et al.,
JCM 5810 2002
7
of S-layer proteins of Gram-positive bacteria are summarized in Table 2.
Table 2. Self-assembly regions in S-layer proteins of Gram-positive bacteria.
Strain S-layer Location of crystallization Reference

protein region (residues / total
residues)
Bacillus anthracis Sap C-terminal (211-814 / 814) Candela et al., 2005
Lysinibacillus SbpA Central (203-1031 /1268)*# Huber et al., 2005
sphaericus CCM 2177
Geobacillus SbsB C-terminal (177-889 / 889) Rüntzler et al., 2004
stearothermophilus
PV72/p2
Geobacillus SbsC Central (258-920 / 1099)* Jarosch et al., 2001
stearothermophilus
ATCC 12980
Lactobacillus SA N-terminal (32-321 / 413) Smit et al., 2001
acidophilus ATCC 4356
Lactobacillus crispatus CbsA N-terminal (32-271 / 410) Antikainen et al., 2002
JCM 5810
* Signal sequence included in the numbering.
# Conclusions drawn from separate N- and C-terminal truncations.
1.1.2.4 Functions
Considering the wide occurrence of S-layers in the microbial world, information
about their functions is still insufficient, and no common function for all S-layers
appears to exist. The functions characterized thus far include the determination and
maintenance of cell shape, various protective functions and actions as a molecular
sieve, as a binding site for large molecules or ions and as a mediator of bacterial
adhesion; the contribution to virulence reported for the S-layers of many pathogens
may result from many of these functions (Sara & Sleytr, 2000). Furthermore, one
S-layer protein thus far, SwmA of a marine Synechococcus strain, has been shown
to be involved in motility (Brahamsha, 1996; McCarren et al., 2005), and for three
S-layer proteins, those of Clostridium difficile, Bacillus anthracis and Lactobacillus
acidophilus, a degradative enzymatic function has been demonstrated (Calabi et
al., 2001; Ahn et al., 2006; Prado Acosta et al., 2008). More specifically, S-layers
may offer protection against mechanical and osmotic stress (Engelhardt, 2007a; b),
radiation (Kotiranta et al., 1999), changes in the environmental pH (Gilmour et al.,
2000), bacteriophages (Howard & Tipper, 1973), bacterial or eukaryotic microbial
predators (Koval & Hynes, 1991; Tarao et al., 2009) or enzymes (Lortal et al., 1992).
They may act as binding sites for exoenzymes (Matuschek et al., 1994; Egelseer et
al., 1995; Peters et al., 1995; Egelseer et al., 1996), immunoglobulins (Phipps & Kay,
1988), porphyrins (Kay et al., 1985) or phages (Howard & Tipper, 1973; Ishiguro
et al., 1984; Fouet, 2009), or catch toxic metals (Pollmann et al., 2006) or calcium
leading to mineral formation (Schultze-Lam et al., 1992). In pathogenic bacteria,
S-layers may contribute to virulence by several mechanisms, including adhesion to
host tissues or cells, antigenic variation, protection from phagocytosis or complement
(reviewed by Kotiranta et al., 2000; Sara & Sleytr, 2000) or by suppression (Wang et
8
al., 2000) or induction (Ausiello et al., 2006) of cytokine secretion. On the other hand,
also the S-layer protein of a health-promoting Lactobacillus strain has been shown
to interact specifically with immune cells, regulating their function through cytokine
induction (Konstantinov et al., 2008).
1.1.2.5 Applications
Applications of S-layers can be divided into two groups. The first comprises
applications utilizing (engineered) S-layered bacterial cells, S-layer (fusion) proteins
or only the expression and/or secretion signals of S-layer protein genes in various
biological systems, including vaccine development, heterologous protein production
and surface display. The second group utilizes isolated, usually recombinant S-layer
proteins for (nano)biotechnological applications. In vaccine development, the high
antigen amount provided by the S-layer (carrier) as well as the intrinsic adjuvant
and immunostimulatory properties of the S-layer arrays (Smith et al., 1993) may be
advantageous (Seegers, 2002; Wells & Mercenier, 2008). As a few examples, S-layer
protein preparations purified from pathogens have been tested as vaccines in fish
(Lund et al., 2003) or in animal models for AAD (antibiotic-associated diarrhoea) (Ni
Eidhin et al., 2008), and S-layers chemically coupled with polysaccharide antigens
have shown potential as therapeutic cancer vaccines or as traditional vaccines in
animal models (reviewed by Sleytr et al., 1999). S-layer-antigen fusion proteins,
either on/in bacterial cells or as isolated proteins, have produced humoral responses
and/or protection against challenge in animals (Mesnage et al., 1999b; Umelo-Njaka
et al., 2001; Riedmann et al., 2003; Liu et al., 2008), and an S-layer-allergen fusion
protein has proved effective in modulating the immune response to a more favourable
one in experiments utilizing immune cells of allergic humans in vitro (Bohle et al.,
2004). As further examples of the first application group, tools for immunoassays
or for bioremediation have been generated by the display of the immunoglobulin
binding domain of Protein G (Nomellini et al., 2007) or a hexahistidine tag (Wang
et al., 2004; Patel et al., 2009), respectively, in the S-layer protein on bacterial cells.
Further, an immunogenic mycobacterial peptide has been efficiently produced in a
biologically active form using S-layer gene expression signals (Salim et al., 1997), and
a plasmid-based secretion system utilizing the secretion signal of the S-layer protein
of Caulobacter crescentus has been developed and commercialized (Bingle et al.,
1997a; 2000; Duncan et al., 2005). The second group of applications is largely based
on the ability of S-layer proteins to spontaneously form periodic, porous structures
on various supports with identical physicochemical properties on each molecular unit
down to the nanometer scale, and this application field is expanded further by the use
of fusion proteins. Several excellent reviews on nanobiotechnological applications
of S-layer proteins are available (Pum & Sleytr, 1999; Sleytr et al., 1999; Schuster
et al., 2006; Sleytr et al., 2007; Schuster & Sleytr, 2009). Some of the conventional
applications in this field include the use of S-layers as ultrafiltration membranes and
as matrices for the covalent attachment of molecules (enzymes, antibodies, protein A,
biotin, avidin, fluorophores) for use in affinity membranes, amperometric or optical
biosensors or solid-phase immunoassays (Pum & Sleytr, 1999; Sleytr et al., 1999; Sleytr
& Beveridge, 1999; Scheicher et al., 2009). More recently, S-layers proteins have been
9
genetically fused to e.g. enzymes, streptavidin, specific antibody fragments, green

fluorescent protein (GFP) and a protein A immunoglobulin-binding domain analogue
(Z domain); these fusion proteins, which retain the ability to recrystallize, may find
numerous applications varying from biosensors and label-free detection systems to
blood detoxification (Schuster et al., 2006; Schäffer et al., 2007; Sleytr et al., 2007;
Tschiggerl et al., 2008). S-layer proteins can also be recrystallized on lipid films and
liposomes, which causes a remarkable stabilization of these structures. S-liposomes
have a broad application potential as drug or plasmid delivery and targeting systems
with a possibility for specific receptor-mediated intake. As S-layer-supported lipid
membranes on porous or solid supports maintain their functionality and allow even
single membrane protein (pore) recordings, they are valuable tools in drug discovery
and protein-ligand screening and have potential as membrane biosensors and in the
development of electronic and optical devices (Schuster et al., 2006; Sleytr et al., 2007;
Schuster & Sleytr, 2009). S-layers have been used in microlithographic procedures
in which patterns are formed on S-layers on solid supports by ultraviolet irradiation
(Pum et al., 1997a;b; Sleytr et al., 1999); the patterned S-layers are currently used as
resistors in electronics (Pum & Sleytr, 1999). Finally, S-layers have been exploited
in the formation of regularly arranged nanoparticles for applications in molecular
electronics and non-linear optics. These applications include wet chemical processes,
i.e. the formation of nanoparticle superlattices on the S-layers in metal-salt solutions,
including processes in which the binding nanoparticles are preformed and thus of
defined size, and systems in which S-layers act as etching masks before the deposition
of the particle-forming metal (Pum & Sleytr, 1999; Sleytr et al., 1999; 2007; Badelt-
Lichtblau et al., 2009). At present, most of the above mentioned applications are,
however, at the stage of invention and development rather than in commercial use.
1.2. Lactobacilli and their S-layer proteins
Lactic acid bacteria are Gram-positive, non-pathogenic micro-organisms characterized

by the production of lactic acid as the main end-product of carbohydrate metabolism.
Besides having a long history of use in food and feed fermentations, lactic acid
bacteria have aroused interest because of the health beneficial (probiotic) properties of
some strains. Probiotic preparations have been shown to be effective in, for example,
the treatment or prevention of rotavirus or antibiotic associated diarrhea, relief of
the symptoms of irritable bowel syndrome, treatment of inflammatory bowel disease
or pouchitis, prevention and treatment of atopic disease and prevention of recurrent
urinary tract infections in women (Pham et al., 2008). Other beneficial effects,
such as beneficial influences on malignancies, on plasma lipid levels or on lactose
maldigestion in humans, have also been suggested (Ouwehand et al., 2002; Ljungh
& Wadström, 2006). Furthermore, lactic acid bacteria are attractive candidates for
biotechnological health-related applications currently under investigation, such as oral
vaccination, passive immunization, tolerance induction or the development of strains
producing pharmaceutically important proteins (enzymes, microbicides, cytokines) in
vivo (Seegers, 2002; Steidler, 2003; Wells & Mercenier, 2008).
10
The genus Lactobacillus forms a large, very heterogeneous group among lactic
acid bacteria, the one most often used in probiotic preparations. It consists of non-
sporulating, anaerobic or microaerophilic, catalase-negative, fermentative organisms
with a low G+C percent (32-53%) and complex nutritional requirements. Lactobacilli
have been isolated from various environments, including plants, foodstuffs, silage
and sewage, and they have been found in the gastrointestinal and genital tracts of
humans and animals, where they form part of the normal flora (Kandler & Weiss,
1986; Axelsson, 1998; Hayashi et al., 2005; Felis & Dellaglio, 2007). According to
recent culture-independent enumerating methods utilizing either tissues or contents
of the gastrointestinal canal of humans, they seem, however, to represent a minor
proportion (0.01-4.9%) of the total microbial flora and part of this may comprise
transients. In contrast, in the human oral cavity, lactobacilli may attain considerable
populations (Walter, 2008), and in the human female urogenital tract they usually
dominate the healthy microbiota (Redondo-Lopez et al., 1990; Zhou et al., 2007).
In animals, lactobacilli are found in the crops (Fuller & Brooker, 1974; Guan et al.,
2003) and ceca (Zhu et al., 2002) of chickens and in the gastrointestinal tracts of
pigs (Fuller et al., 1978; Pedersen & Tannock, 1989; Pryde et al., 1999; Leser et al.,
2002; Konstantinov et al., 2006), horses (Yuki et al., 2000; Bailey et al., 2003; Al
Jassim et al., 2005), ruminants (Krause et al., 2003; Collado & Sanz, 2007; Busconi
et al., 2008) and rodents (Savage et al., 1968; Morotomi et al., 1975; Diaz et al.,
2004; Lesniewska et al., 2006). The Lactobacillus brevis strain ATCC 8287 used in
this study has originally been isolated from green fermented olives. L. brevis is often
detected in the oral cavity and faeces of humans (Walter, 2008), and the strain ATCC
8287 has been shown to survive passage through the human gastrointestinal tract
(Rönkä et al., 2003).
1.2.1 Occurrence and general properties of Lactobacillus S-layer proteins

In the genus Lactobacillus, S-layers have been found in several, but not all, species.
In public databases, sequences of S-layer protein genes from strains of L. brevis, L.
helveticus, L. suntoryeus and organisms of the former L. acidophilus group (Johnson
et al., 1980), including L. acidophilus, L. crispatus and L. gallinarum, are available.
Furthermore, the Apf1 and Apf2 proteins of L. gasseri and L. johnsonii of the same
group, the gene sequences of which are available, have been described as S-layer-
like (Ventura et al., 2002). In addition, strains of L. amylovorus (Boot et al., 1996a),
L. buchneri (Masuda & Kawata, 1981; 1983), L. kefir and L. parakefir (Garrote et
al., 2004) have been shown to possess an S-layer, although the genes have not been
sequenced. S-layers have been demonstrated by electron microscopy also on L.
fermentum and L. delbrueckii subspecies bulgaricus (Kawata et al., 1974; Masuda &
Kawata, 1983), but the species identification of these strains has subsequently been
questioned (Boot et al., 1996a), and at present these species can be considered non-
S-layered. Likewise, in an early study, a regular layer was seen on L. casei (Barker &
Thorne, 1970), but according to Boot et al. (1996b), no S-protein encoding genes are
present in this species, and the isolate probably would now be reclassified to another
species.
11
All of the Lactobacillus S-layer proteins characterized thus far are preceded by a 25-30
amino acid signal peptide indicating secretion through the general secretory pathway.
The deduced amino acid sequences of mature Lactobacillus S-layer proteins vary
considerably, the range of identical amino acids varying from 7% to 100% (Åvall-
Jääskeläinen & Palva, 2005), and even the S-layer proteins of the same strain may
be markedly different in sequence (Jakava-Viljanen et al., 2002; Hagen et al., 2005).
As in the case of S-layers in general, similarity between the deduced amino acid
sequences, when present, can be found only between related species, e.g. between
the S-layer proteins of the former L. acidophilus group organisms and L. helveticus
(Antikainen et al., 2002; Hagen et al., 2005). However, when the phylogenetic trees
constructed on the basis of 16S rRNA or tuf gene sequences of a set of L. acidophilus-
related organisms, including strains of the novel L. suntoryeus species, were compared
with those constructed on the basis of S-layer protein genes of the same species, the
novel strains no longer grouped together, indicating strong selective pressure forcing
the diversification of S-layer protein genes within L. acidophilus-related organisms
as well (Cachat & Priest, 2005). In a similar analysis, however, the comparison of
phylogenetic trees based on 22 deduced Lactobacillus S-layer protein sequences and
16S rRNA sequences of corresponding Lactobacillus species available revealed a
similar overall clustering of strains (Åvall-Jääskeläinen & Palva, 2005).
S-layer proteins of lactobacilli differ from S-layer proteins in general in their
smaller size (25-71 kDa) and a high predicted overall pI value (9.4-10.4). The lattices
formed by Lactobacillus S-layer proteins characterized thus far are of oblique or
hexagonal type (Åvall-Jääskeläinen & Palva, 2005). An electron micrograph of the
S-layer lattice of Lactobacillus brevis is shown in Fig. 2. A glycan structure on a
Figure 2. The self-assembly product of the recombinant S-layer protein of Lactobacillus brevis
ATCC 8287 observed by negative staining and transmission electron microscopy. Bar, 100 nm.
Figure by Ulla Hynönen.
12
Lactobacillus S-layer protein has to date been described only for L. buchneri (Messner
et al., 2008), but glycosylated S-layer proteins have been described also in L. kefir
(Mobili et al., 2009a). As mentioned earlier, secondary structure predictions for S-layer
proteins are of limited value thus far (see Section 1.1). A prediction performed for the
amino acid sequences of the unprocessed forms of six Lactobacillus S-layer proteins
suggested on average 14% α-helices, 39% extended strands and 47% random coil in
these proteins (Åvall-Jääskeläinen & Palva, 2005). In the S-layer-like proteins Apf1
and Apf2 of L. gasseri and L. johnsonii, the β-sheet content was predicted to be 26-31%
and the overall folding of the proteins was suggested to be irregular (Ventura et al.,
2002). Physical measurements revealing secondary structures have been performed
for a few Lactobacillus species. A Fourier transform infrared (FTIR) spectroscopy
study performed for the S-layer proteins of L. kefir and L. brevis indicated α-helix
contents of 0-21%, β-sheet contents of 23-50% and other structure contents, including
β-turns and random coil, of 37-63 % in these proteins. Interestingly, the proportions
of α-helix, β-sheet and other structures in SlpA of L. brevis ATCC 8287 studied in
this thesis work, were 0%, 50% and 50%, respectively (Mobili et al., 2009b). Atomic
force microscopy (AFM) studies of the S-layer protein CbsA of L. crispatus and its N-
and C-terminal fragments suggested the presence of at least four α-helical structures
of variable sizes, rather than β-sheets, in the N-terminal part of CbsA (Verbelen et al.,
2007). Until now, no three-dimensional structures of Lactobacillus S-layer proteins
on atomic resolution have been available.
1.2.2 Expression of Lactobacillus S-layer protein genes

The very efficient synthesis of S-layer proteins in lactobacilli is achieved by several
means: i) The half lives of the S-layer protein gene transcripts of L. brevis (Kahala
et al., 1997) and L. acidophilus (Boot et al., 1996b) have been determined to be
exceptionally long (14 and 15 min, respectively). In the case of L. acidophilus, this is
supposed to be due to the long 5´ untranslated region (UTR) of the transcript forming a
stabilizing secondary structure (Boot et al., 1996b), while the 5´ UTR of L. brevis slpA
transcript is not exceptionally long (Vidgren et al., 1992). ii) A biased codon usage,
correlating with efficient gene expression in lactobacilli (Pouwels & Leunissen, 1994),
has been observed for the S-layer protein genes of L. brevis (Vidgren et al., 1992) and
L. acidophilus (Boot et al., 1995) as well as for the S-layer-like protein genes apf1 and
apf2 of L. gasseri and L. johnsonii (Ventura et al., 2002). iii) The promoters of S-layer
protein genes are efficient, even to the extent that they have been used in heterologous
expression and protein production systems (see Section 1.2.5). In the promoter regions
of the apf1 and apf2 genes of L. johnsonii encoding S-layer-like proteins, a TG motif
upstream of the -10 box, responsible for increased transcriptional activity, has been
identified (Ventura et al., 2002). iv) Two promoters, offering a possibility to enhance
and/or regulate gene expression, have been identified upstream of the slpA gene of
L. brevis ATCC 8287 (Vidgren et al., 1992), slpB and slpD of L. brevis ATCC 14869
(Jakava-Viljanen et al., 2002), slpA of L. acidophilus ATCC 4356 (Boot et al., 1996b)
and the S-layer-like gene apf1 of L. johnsonii (Ventura et al., 2002). Of these, data
about the use of the promoters are available for L. brevis ATCC 8287 (Kahala et al.,
1997) and L. johnsonii (Ventura et al., 2002), in which both of the promoters are used,
13
and for L. acidophilus, in which only the downstream promoter is functional under the
conditions tested (Boot et al., 1996b).
The presence of multiple S-layer protein genes in the same strain is common in
lactobacilli (Boot et al., 1996a; Hagen et al., 2005). In fact, excluding L. helveticus, for
all Lactobacillus species with genetically characterized S-layer proteins and sequences
publicly available, two or more complete S-layer protein genes in the same strain have
been described. Of these, only the S-layer protein genes slpB and slpD of L. brevis
ATCC 14869 (Jakava-Viljanen et al., 2002), slpA and slpX of L. acidophilus NCFM
(or slpB and slpX of the slpA knock-out mutant of L. acidophilus NCFM) (Goh et al.,
2009) as well as the S-layer-like protein genes apf1 and apf2 of L. johnsonii and L.
gasseri (Ventura et al., 2002) have been shown to be expressed simultaneously. Thus,
silent S-layer protein genes, under the conditions tested, are common and represented
by the slpB genes of L. acidophilus ATCC 4356, NCIMB 8607, LMG 11428, LMG
11469 (Boot et al., 1995) and NCFM (Buck et al., 2005), cbsB of L. crispatus JCM
5810 (Sillanpää et al., 2000), SlpNB of L. crispatus LMG 12003 (unpublished,
GenBank AF253044), slpC of L. brevis ATCC 14869 (Jakava-Viljanen et al., 2002),
by several lgs genes of L. gallinarum (Hagen et al., 2005), and probably also by one
of the two S-layer protein genes identified in L. amylovorus by hybridization (Boot
et al., 1996a), although the presence of two identical-sized S-layer proteins on the
bacterial surface cannot be excluded. According to a preliminary SDS-PAGE analysis
of seven porcine L. amylovorus isolates, only one isolate was suggested to express
two S-layer protein genes at the same time, while in the rest only one S-layer protein
was present (Jakava-Viljanen & Palva, 2007). The genomes of L. gallinarum strains
have two genes encoding S-layer proteins: a common one and a strain-specific one,
but each strain produces only a single S-layer protein, which is always encoded by
the strain-specific gene (Hagen et al., 2005). In the recently sequenced genome of
L. brevis ATCC 367 (Makarova et al., 2006), two complete genes and one truncated
S-layer protein gene have been identified by homology, but nothing is known about
the expression of these genes.
The mechanism of the differential expression of slp genes has been well
documented in L. acidophilus 4356, in which an inversion of a chromosomal segment
leads to the placement of the silent gene in front of the active S-promoter. This event
seems to be unfavoured under laboratory conditions, as the silent gene is at the
expression site only in 0.3% of the chromosomes of a broth culture of L. acidophilus
4356. No conditions favouring the expression of the silent gene have thus far been
characterized (Boot et al., 1996). A similar chromosomal inversion mechanism has
subsequently been shown to operate in L. acidophilus NCFM, where the inactivation
of the S-layer protein gene slpA by homologous recombination leads to the appearance
of an alternate S-layer protein, SlpB, in the mutant strain NCK1377-CI (Buck et al.,
2005; Konstantinov et al., 2008).
Information about adaptive changes in Lactobacillus S-layer gene expression, not
known to involve chromosomal rearrangements, is scarce. In L. brevis ATCC 14869,
the differential expression of the slpB and slpD genes is related to the oxygen content
of the growth medium and the growth stage: slpB is expressed irrespective of oxygen
content and equally in different growth phases, while slpD is predominantly expressed
14
in aerated cultures mainly in the exponential phase. The onset of slpD expression is
most likely mediated by a soluble, cytoplasmic factor and it was surmised to be part of
a stress response; a concomitant change in colony morphology, presumably not directly
linked to the S-layer protein type, was also observed. Neither the nature or mechanism
of action of the soluble regulator nor the reason for the silence of the slpC gene in this
strain is known (Jakava-Viljanen & Palva, 2007). Stress-mediated regulation was also
suggested for the expression of the S-layer protein gene of L. acidophilus NCC2628,
which was induced when the strain was cultivated under conditions of limited protein
supply (Schär-Zammaretti et al., 2005).
Although the S-layer protein genes seem to be essential for lactobacilli, as
S-layer-negative mutants are difficult or impossible to create (Boot et al., 1996;
Martinez et al., 2000; Buck et al., 2005), and expression of S-layer protein genes thus
could be anticipated to be constitutive, the examples above indicate that variation and
regulation at the transcriptional and/or transcriptional level also exists. Recently, genes
encoding alternative sigma factors have been identified in the sequenced genomes
of several Lactobacillus species, and numerous potential transcription factor genes
are also present (Azcarate-Peril et al., 2008). However, currently the transcriptional
and translational regulation mechanisms of Lactobacillus S-layer protein genes on a
molecular level are almost totally unexplored.
1.2.3 Cell wall binding and self-assembly regions in Lactobacillus S-layer proteins
Before this thesis work, the two structural regions of S-layer proteins, the region
involved in the attachment of the S-layer subunit to the cell envelope and the region
involved in S-layer assembly, were characterized in the S-layer proteins of only two
Lactobacillus strains: in the SA protein of L. acidophilus ATCC 4356 (Smit et al.,
2001) and in the CbsA protein of L. crispatus JCM 5810 (Antikainen et al., 2002)
(see also Table 1). Both of these organisms belong to the former L. acidophilus group
(Johnson et al., 1980), and the amino acid sequences of their S-layer proteins show
extensive similarity, especially in the C-terminal parts (Smit et al., 2001), suggesting
a conserved function for the C-terminal region. Extending the alignment to the amino
acid sequences of eight mature S-layer proteins of L. acidophilus group organisms
and the closely related L. helveticus (Collins et al., 1991; Felis & Dellaglio, 2007)
also indicates a remarkable conservation of the C-terminal parts (Antikainen et al.,
2002).
In both SA of L. acidophilus ATCC 4356 (Smit et al., 2001) and CbsA of L.
crispatus JCM 5810 (Antikainen et al., 2002), the conserved C-terminal part of the
S-layer protein, approximately 125 amino acids or one-third of the mature amino
acid sequence, is responsible for binding to the cell envelope, and the more variable
N-terminal part for the self-assembly of the S-layer protein monomers to a periodic
S-layer lattice. Both of these proteins have a similar charge distribution with a high
predicted pI in the C-terminal part rich in lysines. Overall, the C-terminal parts of
these proteins consist mainly of hydrophilic amino acid residues and are predicted to
contain β-strands (Smit et al., 2001; Antikainen et al., 2002).
Lactobacillus S-layer proteins do not possess SLH domains. Instead, two
repeated amino acid sequences with homology to the tyrosine/phenylalanine
15
containing carbohydrate-binding motifs of clostridial toxins and streptococcal

glucosyltransferases (Wren, 1991; von Eichel-Streiber et al., 1992) are present in the
cell wall binding regions of SA and CbsA, in the C-terminal parts of the silent S-layer
protein SB of L. acidophilus ATCC 4356 and the S-layer protein of L. helveticus
CNRZ 892, as well as in non-S-layer proteins of lactic acid bacteria known to be
associated with the cell envelope or to agglutinate red blood cells (Smit et al., 2001).
The cell wall receptors for the C-terminal parts of SA and CbsA have been shown to be
teichoic acids (Antikainen et al., 2002; Smit & Pouwels, 2002), and CbsA binds also
to lipoteichoic acids (LTA) isolated from Staphylococcus aureus and Streptococcus
faecalis, but not to the teichuronic acid/polysaccharide fraction of the cell wall of L.
crispatus JCM 5810. Based on the lack of amino acid sequence similarity of CbsA
with other positively charged LTA binding proteins, and binding studies performed
after the oxidation of carbohydrates in LTA showing no effect on binding, the LTA
binding of the C-terminal part of CbsA was suggested to be mediated by electrostatic
interactions involving the lysine residues in the CbsA C-terminal part (Antikainen et
al., 2002). Participation of such electrostatic interactions was not excluded in the case
of the cell wall binding of SA either (Smit et al., 2001). For SA, only one of the two
65 amino acid repeats of the cell wall binding region is necessary for binding, and an
enhancing role for the other repeat has been suggested (Smit & Pouwels, 2002). In the
case of CbsA, no further dissection of the C-terminal part for cell wall binding studies
has been performed.
The self-assembly regions of SA and CbsA have been mapped by studying the
self-assembly properties of truncated recombinant proteins by transmission electron
microscopy (Sillanpää et al., 2000; Smit et al., 2001; Antikainen et al., 2002; Smit
et al., 2002). The fragments comprising the C-terminal two-thirds of SA (residues
1-290 in the mature protein) form a lattice with p2 symmetry, identical to that formed
by SA extracted from L. acidophilus ATCC 4356 cells (Smit et al., 2001). The
lattice parameters or symmetry type of the lattice formed by full-length recombinant
CbsA and its N-terminal self-assembly part (residues 32-271 in the mature protein)
(Antikainen et al., 2002) have not been determined.
Both SA and CbsA can be viewed as two-domain proteins with an N-terminal
domain facing the environment and a non- or less-exposed C-terminal domain; in
SA this view was supported by proteolytic and chemical breakdown experiments
(Smit et al., 2001). According to insertion and deletion mutagenesis and proteolytic
studies of SA, the N-terminal self-assembly domain is probably organized into two
subdomains of approximately 12 and 18 kDa, linked by a surface-exposed loop. The
very N-terminus of SA is not critical for crystallization and is probably buried inside
the domain or facing the cell wall or S-layer pore. Conserved regions and regions
predicted to form secondary structures in SA are necessary for the formation of
a regular lattice (Smit et al., 2002). The lack of necessity of the very N-terminal
end and the importance of the conserved regions for self-assembly have also been
demonstrated for CbsA, where the conserved, valine-rich flanking regions of the self-
assembly domain (residues 30-32 and 269-273 in mature CbsA) have been shown
to be especially important for the formation of the S-layer lattice and may have a
role in directing the formation of a regular polymer: changes in the morphology of
16
the self-assembly products of CbsA fragments are seen accompanying a mutation of

even a single residue in these conserved border regions as well as with the stepwise
truncation of the self-assembly region. Although not necessary for self-assembly, the
C-terminal cell wall binding domain has a stabilizing role in the recrystallization of
CbsA monomers by allowing a more efficient sheet formation. The region in CbsA
responsible for self-assembly also binds collagen (see Section 1.2.4), and the binding
correlates with the ability of recombinant CbsA fragments to form a regular lattice
structure (Antikainen et al., 2002).
In addition to the two well-characterized proteins described above, only
fragmentary data are available about the cell wall binding or self-assembly of other
Lactobacillus S-layer proteins. In early studies, the cell walls of L. buchneri (Masuda
& Kawata, 1981) and L. brevis ATCC 8287 (Shimohashi et al., 1976) were shown to
contain neutral polysaccharides, which were suggested to be involved in the anchoring
of the S-layer protein to the cell wall through hydrogen bonding (Masuda & Kawata,
1980; 1981; 1985). In comparison with the well-characterized exopolysaccharides of
lactic bacteria (De Vuyst & Degeest, 1999; Welman & Maddox, 2003), the cell wall
polysaccharides of lactobacilli other than teichoic acids are poorly known. The detailed
structure of a neutral wall polysaccharide of L. casei has been determined (Nagaoka
et al., 1990), but no precise structures for such polysaccharides of L. buchneri or L.
brevis strains are available. Regarding organisms related to L. acidophilus, the S-layer
protein of L. helveticus CNRZ 892 can, based on amino acid sequence similarity, be
anticipated to be composed of similar functional domains as SA and CbsA, although
the detailed mechanisms of cell wall binding and, especially, self-assembly are more
likely to vary.
1.2.4 Functions of Lactobacillus S-layer proteins

Until now, only a couple of functions have been shown or proposed for Lactobacillus
S-layer proteins. The presence of the S-layer protein decreases the susceptibility of
L. helveticus ATCC 12046 to mutanolysin (Lortal et al., 1992) and the susceptibility
of L. acidophilus M92 to gastric and pancreatic juice (Frece et al., 2005), and a role
as a phage receptor has been suggested for the S-layer protein of L. helveticus CNRZ
892 (Callegari et al., 1998). The auxiliary S-layer component SlpX of L. acidophilus
NCFM probably affects the permeability of the S-layer, as the slpX-negative mutant
is more susceptible to SDS and more resistant to bile than the wild type (Goh et
al., 2009). Recently, the C-terminal part of the S-layer protein SA of L. acidophilus
ATCC 4356 was shown to have murein hydrolase (endopeptidase) activity against the
cell wall of e.g. Salmonella enterica (Prado Acosta et al., 2008), but the biological
relevance of this finding was not investigated.
The most often proposed function for Lactobacillus S-layers is the mediation of
bacterial adherence to various targets. In a number of studies, the loss of the S-layer
protein from the bacterial surface by chemical means (Kos et al., 2003; Garrote
et al., 2004; Frece et al., 2005; Chen et al., 2007; Jakava-Viljanen & Palva, 2007;
Tallon et al., 2007) or the covering of the layer by other molecules during prolonged
cultivation (Schneitz et al., 1993) has been shown to decrease adhesion to different
targets, but the role of the S-layer protein in adherence in these studies has not been
17
directly demonstrated. The haemagglutinating activity of L. acidophilus JCM 1034

and the mucin binding activities of related strains were supposed to be linked to their
S-layer proteins, but the involvement of other guanidine hydrochloride-extractable
components of the cell wall in this lectin-like activity could not be excluded, and
no attention was paid to the aggregation of the S-layer proteins possibly causing
unspecific effects (Yamada et al., 1994; Takahashi et al., 1996). Likewise, in the study
of Golowczyc et al. (2009), where the carbohydrate-dependent co-aggregation of L.
kefir with yeast or red blood cells was suggested to be S-layer-mediated, conclusions
were drawn from the effects of LiCl and SDS treatments of L. kefir cells, and non-
soluble LiCl extracts of L. kefir were used in the aggregation assays. The study of
Uchida et al. (2006), in which the dialysed guanidine hydrochloride extract containing
the S-layer protein of L. brevis OLL2772 was shown to bind to the human blood group
A antigen in a surface plasmon resonance assay, can especially be criticized for the
use of an aggregated solution as an analyte. Inconclusive and indirect evidence of
S-layer protein binding to epithelial cells is also available from studies where dialysed
guanidine hydrochloride- or lithium chloride extracts of L. helveticus (Johnson-
Henry et al., 2007) or L. crispatus (Chen et al., 2007), containing aggregates of the
S-layer proteins of the strains, inhibit the binding of pathogenic E. coli or Salmonella
strains to epithelial cells. In a related study, the lithium chloride-extracted, aggregated
S-layer protein of a L. kefir strain was shown to bind to Salmonella cells, and a role
for the S-layer protein in the inhibition of Caco-2/TC-7 cell association and invasion
of Salmonella by L. kefir was suggested (Golowczyc et al., 2007).
Before this work, the role of a Lactobacillus S-layer protein in bacterial adherence
had been unequivocally shown in two cases, where recombinant S-layer proteins
or S-layer-negative mutants had been used (Toba et al., 1995; Buck et al., 2005;
Konstantinov et al., 2008). First, CbsA of L. crispatus JCM 5810 binds collagen types
I and IV (Toba et al., 1995; Sillanpää et al., 2000). L. crispatus JCM 5810 cells also
bind to collagen-rich regions of chicken colon in vitro, while guanidine hydrochloride-
treated cells are unable to bind, suggesting biological relevance for the observed
collagen binding of CbsA (Sillanpää et al., 2000). The N-terminal amino acids 31-274
of mature CbsA are needed for collagen binding, and practically the same residues
(32-271) are needed for the reassembly of CbsA monomers to an S-layer, suggesting
the dependence of collagen binding on the periodic structure (Sillanpää et al., 2000).
However, the display of CbsA on the surface of a non-S-layered L. casei strain through
a PrtP cell wall anchor renders the recombinant cells able to bind collagens, although
the anchoring system probably does not allow the monomers to form a true S-layer
(Martinez et al., 2000). In contrast, the recombinant form of the non-expressed SlpB
protein of L. crispatus JCM 5810 does not bind collagen types I or IV (Sillanpää
et al., 2000). The second well-characterized adhesive Lactobacillus S-layer protein
is SlpA on L. acidophilus NCFM cells, which binds to the DC-SIGN receptor on
human immature dendritic cells, leading to cytokine production and modulation of
the immune response. The slpA knock-out mutant expressing SlpB is significantly
reduced in binding to DC-SIGN, and the interaction leads to the induction of different
cytokines (Konstantinov et al., 2008). A role for SlpA of L. acidophilus NCFM has
also been demonstrated in binding to Caco-2 cells, as the binding of the knock-out
18
mutant is decreased by 84% compared with the wild type (Buck et al., 2005). The
region in L. acidophilus SlpA responsible for binding to DC-SIGN or Caco-2 cells
has not been determined.
While this thesis work was in progress, a direct interaction between the
chromatographically purified, monomeric form of the S-layer protein SlpA of L. brevis
ATCC 8287 and soluble fibronectin or laminin was demonstrated by surface plasmon
resonance assays. The binding mechanisms to fibronectin and laminin were found to
be different and proposed to be mediated by different regions of SlpA (de Leeuw et
al., 2006). Later, in the study of Khang et al. (2009) SlpA-GFP fusion proteins were
shown to bind to undifferentiated human HT-29 cells, although appropriate controls
were lacking and no attention was paid to the solubility of the protein, making the
specificity questionable.
In addition to the above-mentioned examples of specific binding, the S-layers
of lactobacilli may have a non-specific enhancing effect on binding to surfaces, as
they are generally hydrophobic (see Section 1.1.2.1)(van der Mei et al., 2003). Some
Lactobacillus S-layers, but not all, have even been found to change their surface
hydrophobicity in response to environmental ionic strength, thus possibly offering
different binding capacities. In the case of the SA protein of L. acidophilus ATCC
4356, the decrease in hydrophobicity associated with higher environmental ionic
strength is hypothesized to be due to the shrinkage of the S-layer and the consequent
partial exposure of the inner, more hydrophilic N-terminal domain (see Section 1.2.3)
(Vadillo-Rodriguez et al., 2004).
1.2.5. Applications of Lactobacillus S-layer proteins

Until now, rather few applications have been developed for the S-layer proteins
of lactobacilli. The field currently most extensively studied is the construction of
S-layer fusion proteins for use in immunization in man or animals. Especially, the
development of live Lactobacillus strains carrying S-layers composed of the hybrid
proteins on their surface is of interest, as such strains have potential for use as live
mucosal vaccines. Small model peptides have been displayed in each monomer
of the S-layer of L. brevis ATCC 8287 (Åvall-Jääskeläinen et al., 2002) and L.
acidophilus ATCC 4356 (Smit et al., 2002) by homologous recombination. Similarly,
surface display of green fluorescent protein (GFP) in the S-layer proteins on chicken
Lactobacillus isolates has been achieved by utilizing the gene fragment encompassing
the expression and secretion signals and the region encoding the cell-wall binding
domain of the S-layer protein of L. crispatus (Mota et al., 2006). Furthermore, the
identification of the S-layer protein of L. acidophilus NCFM as the binding ligand for
the dendritic cell-specific antigen DC-SIGN (Konstantinov et al., 2008) makes this
probiotic strain or its S-layer a very attractive tool for oral vaccine design. A system
utilizing L. acidophilus NCFM (but not its S-layer) as a carrier for an antigen with a
small dendritic cell targeting peptide has already been developed (Mohamadzadeh et
al., 2009). Preliminary experiments have also been performed in the field of passive
immunization by utilizing the S-layer protein of L. brevis KCTC 3102 (ATCC 8287) as
a purified, immunoglobulin binding fusion protein to target antibodies to the intestinal
surfaces of calves in order to prevent neonatal diarrhoea (Khang et al., 2009).
19
Another potential application is the use of S-layers or S-layered lactobacilli as

anti-adhesion agents for the prevention of infectious diseases. The collagen, laminin
and fibronectin binding L. crispatus strain JCM 5810 (Toba et al., 1995) and its
well-characterized collagen adhesin CbsA (see Section 1.2.4) inhibit the binding of
enterotoxigenic E. coli to a reconstituted basement membrane preparation as well
as to its main component laminin in vitro (Horie et al., 2002). The observed anti-
adhesive effects of lactobacilli or their surface protein extracts mentioned in Section
1.2.4., including those of L. crispatus (Chen et al., 2007), L. helveticus (Sherman et
al., 2005; Johnson-Henry et al., 2007) and L. kefir (Golowczyc et al., 2007) have been
presented as useful, probiotic traits, although the extracts used in the inhibition studies
apparently contained aggregates of the S-layer protein, and thus, the specificities of
the inhibitions were compromised. In addition, the experiments have thus far only
been performed in vitro, and their application potential in human or animal medicine
is obscure.
The biochemical modification studies of Lactobacillus S-layer proteins for
technological applications have recently been initiated, with small molecular probes
like biotin or FITC conjugated to purified S-layer proteins of L. brevis using amine-
based coupling chemistry. The S-layer protein bioconjugates formed, purified by
affinity chromatography, were capable of self-assembling into regular layers, where
the surface coverage of the conjugated molecules is homogeneous and the density
controllable. The method offers a way to display several different and high-molecular
weight molecules at an interface (Sampathkumar & Gilchrist, 2004).
The expression and/or secretion signals of Lactobacillus S-layer protein genes
have also been utilized in biotechnological applications (Savijoki et al., 1997; Kahala
& Palva, 1999; Novotny et al., 2005). The signals of the slpA gene of L. brevis ATCC
8287 have been used in Lactobacillus and Lactococcus hosts for intracellular (Kahala
& Palva, 1999) and extracellular (Savijoki et al., 1997) protein production. Using slpA
expression and secretion signals, secretion levels of beta-lactamase up to 80 mg/l have
been achieved. Differences exist between the recognition efficiency of the signals
in different hosts: high-level protein production with slpA signals is achieved in
Lactococcus lactis and Lactobacillus plantarum and moderate in Lactobacillus gasseri,
while in Lactobacillus casei the expression signals are not recognized (Savijoki et al.,
1997). On the other hand, the promoter region of Lactobacillus acidophilus ATCC
4356 S-layer protein gene is highly functional in L. casei (Boot et al., 1996b).
1.2.6. Tools to study S-layer structure and function: cysteine scanning mutagenesis
and bacterial surface display
The typical features of S-layer proteins, the rather large molecular weight and poor
solubility (see Section 1.1.2.1) pose problems for the structural and adhesion studies
of also Lactobacillus S-layer proteins, necessitating the use of methods overcoming
these difficulties, like cysteine scanning mutagenesis or flagellar display.
1.2.6.1. Cysteine scanning mutagenesis and sulfhydryl chemistry

CSM is based on the targeted mutagenesis of selected amino acid residues in the protein
of interest to cysteines, which are then amenable to chemical modification owing to
their sulfhydryl groups. The stability and functionality of the mutant proteins generated
20
are first tested. Several application modes can then be used. In chemical reactivity
scanning, only one residue in each mutant protein is changed, resulting in a series of
mutant proteins each having a single modifiable residue at a different location. Several
sulfhydryl-reactive reagents can be used for the modification of these residues, and
the modified proteins can be distinguished from the unmodified ones by, for instance,
fluorescence or a change in molecular weight. The extent of modification is a measure
of the surface accessibility of the mutated residue. In disulphide-based applications,
one or a pair of cysteine residues is introduced to the protein, and the engineered
sulfhydryl groups at different locations in the generated pool of mutant proteins are
allowed to react with each other. The presence of disulphide bonds is detected based
on migration in a polyacrylamide gel, and the observed contacts between pairs of
positions elucidate the tertiary or quaternary structure of the protein (Bass et al.,
2007). Disulphide mapping as a method of investigating the proximity of two residues
in a protein is thus based on the same principle as chemical cross-linking, also used in
structural and interaction studies (Kluger & Alagic, 2004; Sinz, 2006), although the
distance constraints between the reacting residues may be different. A limitation of
CSM is that the wild-type protein is not allowed to contain cysteines, but in S-layer
proteins this does not generally pose a problem, as cysteines in bacterial S-layer
proteins are usually absent (see Section 1.1.2.1). Major advantages of the method are
that it can be applied to an entire protein or to a specific domain or subdomain, and it
can be carried out in the native environment of the protein, e.g. in a lipid bilayer or a
multiprotein complex, making it a method of choice for many proteins inaccessible
to high-resolution methods like X-ray crystallography. Furthermore, it is a versatile
system that is able to map out secondary, tertiary and quaternary structures, analyse
conformational changes and structure-function relationships and reveal thermal
motions within a protein structure by a method called disulphide trapping (Frillingos et
al., 1998; Bass et al., 2007). However, CSM-based methods are all rather laborious.
CSM has been widely used in the study of membrane proteins and large
heteromeric multiprotein complexes of both bacterial and eukaryotic proteins (Bass
et al., 2007). Especially, the structure and function of bacteriorhodopsin (Altenbach
et al., 1990), microbial pore-forming toxins (Merzlyak et al., 2005; Iacovache et al.,
2006; Girard et al., 2008), microbial transporters of drugs, antibiotics, nutrients or
ions (Sobczak & Lolkema, 2005; Hassan et al., 2006; Kuwabara et al., 2006; Xu
et al., 2006; Greene et al., 2007; Papakostas et al., 2008; Wang et al., 2008) and
microbial membrane components involved in environmental sensing (Goldberg et al.,
2008), chemo- and aerotaxis (Winston et al., 2005; Bass et al., 2007; Taylor et al.,
2007) and protein secretion (Mori et al., 2004) have been studied by CSM. Before this
thesis work, the only CSM study of a bacterial S-layer protein had been performed
with the SbsB protein of Geobacillus stearothermophilus PV72/p2. In that work,
the spatial locations of 75 residues out of 920 were studied, and 23 of them were
found to be located on the surface of SbsB monomers (Howorka et al., 2000; Kinns &
Howorka, 2008). In a subsequent study, these selected mutant proteins with surface-
accessible cysteines were analysed by chemical cross-linking to find the residues
located at the protein/protein interface or on the inner surface of the S-layer lattice.
Specific modified sites were also found to be subject to intramolecular cross-linking,
indicating conformational changes upon self-assembly (Kinns & Howorka, 2008).
21
1.2.6.2 Flagellar display

Bacterial surface display means the presentation of foreign peptides on the bacterial
surface, usually through recombinant DNA technology. Both Gram-positive and Gram-
negative bacterial species have been used as display hosts (Samuelson et al., 2002).
In Gram-positive bacteria, the most common anchoring mechanism of the foreign
peptide to the cell envelope is a covalent linkage to peptidoglycan through the LPXTG
sequence of the foreign peptide, but other anchoring peptide sequences derived from
Gram-positive bacteria (e.g. SLH domains, AcmA repeats) and transmembrane and
lipoprotein membrane anchors have also been used (reviewed by Sillanpää, 2001;
Samuelson et al., 2002; Lee et al., 2003). Studies aiming at the utilization of whole
S-layer proteins (Smit et al., 2002; Åvall-Jääskeläinen et al., 2002; Mota et al.,
2006) (see Section 1.2.5), spores (reviewed by Kim & Schumann, 2009) or flagella
(Crampton et al., 2007) of Gram-positive bacteria as display vehicles have also been
initiated. In Gram-negative bacteria, surface display is based on S-layer proteins
(Bingle et al., 1997b; Nomellini et al., 2007), outer membrane proteins (OMPs) or
OMP-lipoprotein fusions (reviewed by Georgiou et al., 1997; Lång, 2000; Samuelson
et al., 2002), outer membrane proteins modified by circular permutation (Rice et
al., 2006), autotransporters (reviewed by Jose, 2006; Jose & Meyer, 2007), secreted
proteins (Kornacker & Pugsley, 1990), purified intracellular structures like bacterial
magnetic particles (BMPs)(reviewed by Wu et al., 2008) or surface organelles, i.e.
fimbriae (reviewed by Klemm & Schembri, 2000), flagella (reviewed by Westerlund-
Wikström, 2000) or other filaments (Crepin et al., 2005). Bacterial surface display
techniques not involving gene fusions have also been developed for both Gram-
positive and Gram-negative bacteria (Sadamoto et al., 2004; Tanaka et al., 2004;
Bosma et al., 2006).
In the flagellar display system of Gram-negative bacteria, the sequence encoding
the foreign peptide is inserted into the flagellin gene (fliC) encoding the main subunit of
the flagellar filament. Methods have been developed for the flagella of both E. coli and
Salmonella, the flagellar structures of which are very similar (see Fig. 3 for a schematic
presentation). In addition to the helical filament consisting of several thousand copies
of the main subunit FliC, the flagellum is composed of a pentameric cap protein (FliD)
at the tip of the filament, and the hook (FlgE) and the junction proteins (FlgK, FlgL)
between the filament and the basal body structure, which connects the filament to the
outer membrane (Fernandez & Berenguer, 2000; Westerlund-Wikström, 2000). FliC
is composed of four domains: two conserved ones, inner and outer, important for the
polymerization of flagellins and the stability of the flagellum, and a variable surface-
exposed region, composed of two domains, responsible for the antigenic variation of
flagella (Westerlund-Wikström, 2000; Yonekura et al., 2003; Beatson et al., 2006).
The variable region is utilized in the flagellar display system, as it allows the deletion
of up to 187 residues without a loss of flagellar integrity or function (Kuwajima,
1988; Lu et al., 1995). After the replacement of a part of this non-essential sequence
of fliC by the desired sequence, the plasmid construct is used to complement a ΔfliC
host strain, resulting in flagella composed of only the hybrid flagellins (Westerlund-
Wikström et al., 1997; Westerlund-Wikström, 2000).
Flagellar display has been used e.g. for the study of adhesive peptides
(Westerlund-Wikström et al., 1997), for the selection of metal-binding peptides for
22
use as bioadsorbents (Dong et al., 2006) and for vaccination trials (Chauhan et al.,
2005; reviewed by Westerlund-Wikström, 2000; Ben-Yedidia & Arnon, 2005). An
application utilizing FliC- thioredoxin fusions, with random peptides displayed in the
thioredoxin part of the fusion, has also been developed as an alternative to phage display
(Lu et al., 1995). Peptides up to 302 amino acids have been successfully expressed
using flagellar display (Westerlund-Wikström et al., 1997). A limitation of the method
is that the type III system used for the secretion of flagellins bypasses the periplasmic
space, where disulphide bonds are formed (Macnab, 2004), and thus, disulphide bonds
cannot be formed in the peptide to be displayed. In the case of S-layer proteins this,
however, does not usually pose a problem (see Section 1.1.2.1). Flagellar display
has several significant advantages. Hybrid flagella can be used either attached to the
bacterial cell or in purified form, and purification is easily accomplished on a large
scale (Westerlund-Wikström, 2000). In vaccine design, the presence of the antigenic
epitope as multiple copies, the possible adjuvant effects of other accompanying
bacterial components and the capacity of the conserved regions of flagellin to interact
with TLR5 leading to immunostimulation (Eaves-Pyles et al., 2001; Hayashi et al.,
2001) are advantageous (Seegers, 2002; Wells & Mercenier, 2008). The flagellin part
in antigen-flagellin fusion proteins has been shown to induce APC (antigen presenting
cell) maturation, cytokine secretion and the development of strong and specific
immune responses towards the antigen (Cuadros et al., 2004). Flagellins have also been
demonstrated to have a probiotic effect by inducing the synthesis of an antimicrobial
peptide in epithelial cells (Ogushi et al., 2001; Schlee et al., 2007). The multivalency
of the hybrid flagella facilitates their use also in diagnostics and as tools to identify and
isolate adhesins and their receptors (Westerlund-Wikström, 2000). Further versatility
is provided by the possibility to present two or more epitopes simultaneously on the
same flagellar filament by utilizing FliD as well as FliC as fusion partners for the
foreign peptides (Tanskanen et al., 2000; Majander et al., 2005).
Figure 3. Schematic presentation of (A) the flagellar filament and (B) the cross-section of a
Salmonella flagellar filament. C1 and C2, conserved domains; V, variable region composed
of two domains. This figure is a reprint from Westerlund-Wikström (2000), with permission
granted by Elsevier.
23
1.2.7 Non-S-layer adhesive surface proteins of lactobacilli

Despite some contradictory opinions about the importance of Lactobacillus adherence
for persistence in the human gastrointestinal tract (Walter, 2008), the conventional
view is that a good adherence capacity of a strain to mucus or epithelial cells promotes
its colonization or residence time in the gut. As such, this is usually also considered
a desirable trait for a probiotic organism, as it may facilitate pathogen exclusion,
immune modulation and epithelium protection effects possibly exerted by the strain
(Lebeer et al., 2008). Adhesion of lactobacilli has mostly been studied by in vitro
experiments using immortalized cell lines, isolated epithelial cells or tissue fragments,
purified proteins or other components of the extracellular matrix, and mucus or mucus
components as binding targets. Red blood cell, yeast cell and platelet agglutination
assays have also been used. The molecular mechanisms mediating the adherence
remain rather poorly known. A role for carbohydrates on the bacterial surface in
adherence has been proposed in many studies, but only the binding of one non-protein
molecule, LTA, to epithelial cells has been directly demonstrated (Chan et al., 1985) as
well as suggested by inhibition studies (Granato et al., 1999; Kapczynski et al., 2000).
Although not completely excluding the involvement of bacterial carbohydrates, in
many cases Lactobacillus surface proteins seem to mediate adherence.
Considering the large number of studies, in which protein-mediated adhesion
of lactobacilli to various targets, based on the effect of protease treatment, has been
described, relatively few adhesive surface proteins have been functionally and
genetically characterized. On the other hand, in August 2009, 16 complete chromosome
sequences representing 12 Lactobacillus species were publicly available, and these and
ongoing sequencing projects of Lactobacillus genomes are continuously providing
sequence homology-based information about genes potentially involved in adhesion.
For example, the genome of L. plantarum WCFS1 is predicted to encode 12 adhesive
proteins with domains with affinity to mucus, collagen, fibronectin or chitin (Boekhorst
et al., 2006b), and the genome of L. gasseri ATCC 33323, according to an in silico
analysis, encodes as many as 14 proteins with mucus-binding domains as well as
proteins with similarity to bacterial adhesins binding to fibronectin, human epithelial
cells or saliva (Azcarate-Peril et al., 2008). In this section, however, the emphasis will
be on genetically and functionally well-characterized adhesive Lactobacillus surface
proteins.
The role of Lactobacillus S-layer proteins as adhesins has been discussed in
Section 1.2.4. Grouping of the rest of the adhesive proteins according to species or
binding target is difficult since strains of the same species, or even a single strain,
may carry different adhesive proteins and several targets may have been identified
for the same adhesin. In the following, adhesins with amino acid sequence similarity
are presented as groups and an approximate chronological order of identification is
followed. A summary of Lactobacillus non-S-layer adhesins is presented in Table 3.
The first Lactobacillus adhesin characterized at a genetic level was the collagen-
binding protein CnBP of L. reuteri NCIB 11951, which shares amino acid sequence
similarity with the solute binding components of bacterial ABC transporters (Aleljung
et al., 1994; Roos et al., 1996). A homologue of CnBP in L. reuteri 104R, with 94%
amino acid identity (Miyoshi et al., 2006), was originally characterized as a mucus-
24
Table 3. Genetically and functionally characterized non-S-layer adhesive surface

proteins of lactobacilli. FnBp, fibronectin-binding protein; MBp, mucin-binding
protein.
Adhesin Organism Binding target Reference
CnBP L. reuteri NCIB 11951 Collagen Aleljung et al., 1994;
Roos et al., 1996
MAPP/MapA L. reuteri 104R Mucus, Caco-2 cells Rojas et al., 2002;
Miyoshi et al., 2006
32-Mmubp L. fermentum BCS87 Mucus, mucin Macias-Rodriguez et
al., 2009
Mub L. reuteri 1063 Mucus Roos & Jonsson, 2002
LBA 1392 (MBp L. acidophilus NCFM Caco-2 cells Buck et al., 2005
homologue)
Msa L. plantarum WCFS1 Mannosides Adlerberth et al., 1996;
Pretzer et al., 2005
Lsp L. reuteri 100-23 Mouse forestomach Walter et al., 2005
epithelium
LspA L. salivarius UCC118 Caco-2, HT-29 cells van Pijkeren et al., 2006
LBA 1148 (FnBp L. acidophilus NCFM Caco-2 cells Buck et al., 2005
homologue)
EF-Tu L. johnsonii NCC533 Caco-2, HT-29 cells, Granato et al., 2004
mucin
GroEL L. johnsonii NCC533 HT-29 cells, mucin Bergonzelli et al., 2006
Eno L. crispatus ST1 Laminin, collagen Antikainen et al., 2007a
Eno1-3 L. johnsonii F133 Laminin Antikainen et al., 2007a
EnoA1 L. plantarum LM3 Fibronectin Castaldo et al., 2009
GAPDH L. plantarum LA318 Mucin Kinoshita et al., 2008b
adhesion-promoting protein (MAPP) (Rojas et al., 2002), but was subsequently found
to bind to Caco-2 cells as well, and was named MapA (Miyoshi et al., 2006). Another
homologue of CnBP, with approximately 90% amino acid sequence identity, present
as the major constituent of the LiCl extract of L. fermentum BR11, was initially defined
as a basic surface protein (BspA) (Turner et al., 1997). It was later shown to be an
L-cystine binding protein in an ABC transporter system (Turner et al., 1999; Hung et
al., 2005), necessary for oxidative defence (Hung et al., 2003), and the designation
CyuC was suggested (Hung et al., 2005). For BspA/CyuC, no role in bacterial adhesion
has thus far been demonstrated, although it cannot be ruled out, as many bacterial
adhesins are multifunctional (Kukkonen & Korhonen, 2004; Leon-Kempis Mdel et
al., 2006; Antikainen et al., 2007a; Sanchez et al., 2008) and numerous potential
binding targets exist. A third homologue of CnBP (or CnBP itself, deduced from the
20 identical N-terminal amino acids), referred to originally as p29, is present in the
pool of secreted biosurfactants of L. fermentum RC-14 and has anti-adhesive activity
towards E. faecalis (Heinemann et al., 2000). The same protein has subsequently been
shown by SELDI-TOF (surface-enhanced laser desorption/ionization-time of flight)
analysis to bind collagen types III and VI, and its gene has been cloned (Howard et
al., 2000), although no report about the gene sequence is available. Recently, in L.
reuteri JCM 1081, an HT-29 cell- and mucin-binding homolog of CnBP has been
identified (Wang et al., 2008), and an ABC transporter component, which binds to pig
mucus and mucin, has been detected on the surface of L. fermentum BCS87 (Macias-
Rodriguez et al., 2009).
25
The mucus-binding protein Mub of L. reuteri 1063 is one of the largest bacterial
surface proteins (3269 amino acids, 358 kDa) and the first Lactobacillus adhesin shown
to have a LPXTG cell wall anchoring motif. Its 14 so-called Mub-repeats, 200 amino
acids each, are responsible for the binding of the protein to the carbohydrate structures
in pig and hen mucus. Two types of repeats can be distinguished, with slightly different
patterns of inhibition of binding by carbohydrates, but both repeat types are important
for adhesion (Roos & Jonsson, 2002). Based on colony hybridization, the mub gene is
frequently present in pig intestinal Lactobacillus isolates closely related to L. reuteri,
L. fermentum and L. pontis, and these strains have been suggested to constitute a new
species, L. mucosae (Roos et al., 2000).
Several Mub-like proteins in lactobacilli have been functionally characterized. In
L. fermentum BR11, a vaginal isolate of a guinea pig, a large Mub-like protein (Mlp)
has been identified with 10 repeat sequences and 21% overall amino acid sequence
identity with Mub of L. reuteri, but the protein does not bind to pig gastric mucin
(Turner et al., 2003). A Mub homologue, LBA 1392, with 24% overall amino acid
sequence identity has been identified in L. acidophilus NCFM, where it has been shown
by knock-out mutation of the gene to be involved in adherence to Caco-2 cells (Buck
et al., 2005). A Mub-repeat-carrying protein in L. plantarum was initially identified as
a mannose-specific adhesin capable of agglutinating yeast cells and binding to HT-29
cells (Adlerberth et al., 1996). When the whole genome sequence of L. plantarum had
become available (Kleerebezem et al., 2003), the corresponding gene was identified
and cloned, designated msa, and shown by knock-out mutation and complementation
to be responsible for the yeast cell agglutination phenotype of L. plantarum. However,
in addition to Mub-repeats, Msa has a domain with similarity to a ConA-like lectin
(Pretzer et al., 2005). In the in situ pig small intestinal segment perfusion model, Msa
has been shown to contribute to adherence to porcine jejunal epithelium and to induce
host responses (Gross et al., 2008). The large surface protein (Lsp) of L. reuteri 100-
23, which has a role in the adherence of the strain to mouse forestomach epithelium in
vivo and an effect on the colonization dynamics in mice, has 83% nucleotide sequence
identity with a Mub-domain-carrying protein of L. johnsonii (Walter et al., 2005),
and thus, probably has Mub-repeats, although a detailed analysis of the sequence of
Lsp has not been presented. However, in the homologous protein in L. johnsonii,
other domains with predicted adhesive properties are also present. The Mub-repeat-
containing protein LspA of L. salivarius UCC 118 mediates adherence to HT-29 and
Caco-2 cells, as shown by an isogenic LspA negative mutant strain (van Pijkeren
et al., 2006). With the appearance of the whole genome sequences of lactobacilli,
several copies of mub-homologous genes have been identified in L. gasseri (fourteen),
L. johnsonii (nine), L. plantarum (four), L. acidophilus (twelve) and L. brevis, L.
fermentum and L. reuteri (two in each) (Boekhorst et al., 2006a; Azcarate-Peril et
al., 2008). Interestingly, one truncated mub-repeat-containing gene is present in the
plasmid carried by L. reuteri ATCC 55730, raising the question of lateral transfer of
mub genes in lactobacilli (Bath et al., 2005). The presence of a potential adhesin gene
in a Lactobacillus plasmid has been confirmed by others as well (Desmond et al.,
2005).
26
The only functionally characterized Lactobacillus adhesive protein with the procaryotic
fibronectin-binding domain is present in L. acidophilus NCFM. The inactivation of the
gene encoding this protein (LBA 1148) results in a marked decrease in the adherence
of L. acidophilus NCFM to Caco-2 cells (Buck et al., 2005), but the binding of the
strain to fibronectin has not been studied.
One group of Lactobacillus adhesive surface proteins consists of those with a
previously characterized cytoplasmic function (so called moonlighting or anchorless
multifunctional proteins (Sanchez et al., 2008)). The L. johnsonii proteins EF-Tu, a
guanosine-nucleotide binding protein participating in protein synthesis, and GroEL, a
stress-induced chaperonin (heat shock protein), are both present on the bacterial surface
non-covalently bound in addition to their known cytoplasmic locations, and have a
pH-dependent adhesive function. EF-Tu binds to Caco-2 cells, to non-differentiated
HT29 cells from the human colon and to mucins purified from HT29-MTX cells or
from the human colon at low pH, while GroEL has been reported to bind to non-
differentiated HT29 cells and to mucins purified from HT29-MTX cells under the
same conditions (Granato et al., 2004; Bergonzelli et al., 2006). The binding of EF-Tu
to mucus or cells has been proposed to have a role in gut homeostasis (Granato et al.,
2004), but for the binding of GroEL a minor role has been suggested; instead, it may
exert its action by specifically aggregating Helicobacter pylori cells after its liberation
from deceased L. johnsonii cells in the acidic stomach (Bergonzelli et al., 2006).
Another example of a cytoplasmic protein acting as an adhesin on lactobacilli is the
glycolytic enzyme enolase, present on the surface of L. johnsonii F133, L. crispatus
ST1 and L. plantarum LM3. L. johnsonii and L. crispatus enolases bind to laminin
and/or collagen in vitro, and also bind and activate plasminogen, the precursor of the
proteolytic enzyme plasmin present in plasma and extracellular fluids (Antikainen
et al., 2007a), while the EnoA1 of L. plantarum binds fibronectin (Castaldo et al.,
2009). Variable laminin/collagen binding and plasminogen activation efficiencies are
observed in individual enolases of lactobacilli; in L. johnsonii F133, for instance, three
enolase genes are present, of which eno2 is silent under standard laboratory conditions
(Antikainen et al., 2007a). The enolase of L. crispatus binds electrostatically to LTA
and is released from the surface of the bacterium at neutral or basic pH, above its
isoelectric point (Antikainen et al., 2007b). The cell surface-bound form of another
glycolytic enzyme, GAPDH, has been identified as a mucus binding adhesin on L.
plantarum (Kinoshita et al., 2008b), and a putative receptor polysaccharide structure
has been determined (Kinoshita et al., 2008a).
A very distinct group of adhesins in lactobacilli are fimbriae (pili). Fimbriae are
common and well-characterized adhesive organelles in Gram-negative bacteria, but
only recently have they been thoroughly studied in Gram-positive bacteria. After the
initial observation of fimbriae on Corynebacterium renale by electron microscopy
(Yanagawa et al., 1968), fimbriae have been identified in several other Gram-
positive species, including Actinomyces, Ruminococcus, Enterococcus, Clostridium,
Mycobacterium and several species of Streptococcus. A peculiar feature of
Gram-positive fimbriae characterized thus far is that they are covalently linked
to peptidoglycan and their main subunits also to each other, and the subunits are
further stabilized by intramolecular covalent bonds (Proft & Baker, 2009). In the
27
pilus assembly process, a pilin-specific sortase is involved (Ton-That & Schneewind,

2003). For a long time, the presence of fimbriae in lactobacilli was debatable, and, in
August 2009, unequivocal evidence of their role in adherence is still lacking. In early
studies, fimbrium-like protruding structures were detected by electron microscopy
on the surface of L. fermentum (Barrow et al., 1980) and on several human vaginal
isolates belonging to species L. rhamnosus, L. acidophilus, L. jensenii, L. casei and
L. fermentum (McGroarty, 1994), and a role in adherence for these fimbrium-like
structures was suggested. Recently, in the completely sequenced chromosome of
L. johnsonii NCC533, two fimbrial operons were identified, which possibly encode
fimbriae with a glycosylated fimbrial protein reminiscent of the Fap1 fimbrial
adhesin of Streptococcus parasanguis, and fimbriae were also detected by electron
microscopy on L. johnsonii cells (Pridmore et al., 2004), but no functional studies of
these structures on L. johnsonii have been performed. Very recently, in the context of
creating an exopolysaccharide-negative mutant of the probiotic strain L. rhamnosus
GG, fimbrium-like structures were detected by electron microscopy, and the increased
adhesion to mucus and Caco-2 cells of the mutant was postulated to be due to the
more efficient exposure of these structures (Lebeer et al., 2009).
In conclusion, increasing numbers of adhesive Lactobacillus surface proteins are
being characterized as the combined result of comparative genomics and functional
studies, and the general view is that in most cases many factors contribute to the
adherence of a particular strain. The specific receptors in epithelial cells or mucus
are mostly uncharacterized to date. Furthermore, excluding the report of Lsp of L.
reuteri 100-23 (Walter et al., 2005) and that of Msa of L. plantarum (Gross et al.,
2008), studies on the biological role of Lactobacillus adhesive proteins in vivo have
not yet been performed. In the future, more adhesive S-layer proteins will potentially
be discovered.
28
Aims of the Study
2. AIMS OF THE STUDY
As food-grade organisms able to survive, persist and potentially exert health-promoting

effects in the gastrointestinal tract, lactobacilli have aroused interest as possible
carriers of oral vaccine antigens or other medically important molecules. In addition,
the S-layer structure carried by several Lactobacillus species offers an effective means
of expressing foreign peptides as multiple copies in an ordered manner on the bacterial
surface. The goal of this study was to provide comprehensive knowledge about the
structure and function of SlpA of L. brevis and about the expression of its gene in
order to develop SlpA-based tools for live mucosal vaccines or other health-related
applications in man or animals as well as for nanobiotechnology.
Specific aims were as follows:

I To specify the role of the S-layer protein in the adherence of L. brevis ATCC
8287 to human epithelial cell lines and fibronectin
II To identify and characterize the domains responsible for self-assembly and cell
wall binding in the S-layer protein of L. brevis ATCC 8287
III To perform a detailed mapping of individual residues in SlpA to gain further
insight into the structure of SlpA and the S-layer formed
IV To study the function of the two consecutive promoters of the slpA gene
29
Materials and Methods
3. MATERIALS AND METHODS
The bacterial strains, cell lines and plasmids used in Studies I-IV are listed in Table 4.
The methods are described in detail in the original articles and summarized in Table
5.
Table 4. Bacterial strains, plasmids and cell lines used in Studies I-IV
Strain, plasmid or cell Origin / relevant Study Reference or source

line property
Bacterial strains
Lactobacillus brevis Green olives I, II, III, IV ATCC
ATCC 8287
Lactobacillus Human pharynx II ATCC
acidophilus ATCC 4356
Lactobacillus helveticus Industrial starter strain IV Valio, Finland
53/7
Lactococcus lactis MG Plasmid- and prophage- IV Gasson, 1983
1363 free derivative of wt S.
lactis NCDO 712
Escherichia coli JT1 E. coli C600 fliC::Tn10 I Westerlund-Wikström
fimA::cat et al., 1997
Escherichia coli Host for pQE-30 I Stratagene
M15(pREP4)
Escherichia coli DH5αF’ Cloning host for pET-28 II Woodcock et al., 1989
vectors
Escherichia coli Expression host for pET- II, III Novagen
BL21(DE3) 28 vectors
Escherichia coli XL-1 Cloning host in site- III Stratagene
Blue directed mutagenesis
Plasmids
pFliCH7Δ pBluescript II KS(+) I Westerlund-Wikström
with serotype H7 fliC et al., 1997
gene with 174 bp
deletion
pQE-30 Expression vector for I Qiagen
his-tagged constructs
pET-28a(+) Expression vector for II Novagen
pET-28b(+) Expression vector for II Novagen
pKTH5199 pET-28a(+) with slpA III II
and N-terminal his-tag
pKTH2121 pGK12 derivative IV Savijoki et al., 1997
pKTH2095 with P1-
P2slpA, SSslpA and bla-tslpA
Cell lines
Intestine 407 Human embryonic I ATCC (CCL6)
intestine
Caco-2 Human colon carcinoma, I ATCC (HTB-37)
differentiatiates to
enterocyte-like
EA-hy926 Hybrid cell line with I Edgell et al., 1983
characteristics of human
endothelial cells
T24 Human urinary bladder I ATCC (HTB-4)
30
Materials and Methods
Table 5. Methods used in Studies I-IV
Method Used and

described in Study
Genetic methods
Cloning to pFliCH7Δ vector I
Cloning to pQE-30 vector I
Cloning to pET-28 vectors II
Cloning to pKTH2121 vector IV
Site-directed mutagenesis III
Isolation of RNA IV
Northern hybridization IV
TRAC IV
DNA sequencing I, II, III, IV
Immunological methods
Western hybridization I
Immunoelectron microscopy I
Immunofluorescence microscopy I
Adhesion assays with bacteria/flagella
Adherence of bacterial cells to human cell lines and fibronectin I
Haemagglutination by bacterial cells I
Adherence of chimeric flagella to human cell lines and fibronectin I
Bacterial cell wall-related techniques
FITC-labelling of bacterial cells I
Isolation of cell walls of L. brevis II, III
TCA extraction of cell walls II
Determination of total phosphorous II
Protein assays
Expression and purification of his-tagged peptides I, II, III
Extraction of S-layer proteins I, II
Binding of recombinant S-layer proteins to bacterial cells and cell II
walls
Reassembly of recombinant S-layer proteins by dialysis II, III
SDS-PAGE I, II, III, IV
Proteolytic degradation of SlpA II
N-terminal sequencing II
Peptide mapping II
Testing of solvent accessibility of cysteine residues in mutant proteins III
SAXS III
Determination of beta-lactamase activity IV
Determination of aminopeptidase activity IV
Electron microscopy I, II, III
Amino acid and/or nucleotide sequence analyses I, II, III, IV
31
Results and Discussion
4. RESULTS AND DISCUSSION
4.1. Structure and function of SlpA
4.1.1 Adhesive functions (I)

When Study I was initiated, the probiotic effects and potential health-related
applications of Lactobacillus strains were of special interest, and the adherence to host
tissues or cells was rather unanimously considered an important feature of a probiotic
strain. The initial finding that the chemical removal of the S-layer protein from the
surface of L. brevis ATCC 8287 abolishes the binding of the strain to cultured human
epithelial cells prompted us to examine the role of the S-layer protein in adherence to
human cells. As the 435-amino-acid S-layer protein of L. brevis ATCC 8287, SlpA,
was genetically well-characterized (Vidgren et al., 1992), the difficulties in creating
S-layer-negative Lactobacillus mutants (Boot et al., 1996; Martinez et al., 2000; Buck
et al., 2005) and those related to the poor solubility of S-layer proteins were overcome
by utilizing the previously established flagellar display system (Westerlund-Wikström
et al., 1997).
Different regions of the slpA gene were expressed as internal in-frame fusions in
the variable region of the flagellin gene of E. coli, and the adhesive properties of the
resulting chimeric flagella were examined (Figures 1 and 4 and Table I in I). Chimeric
flagella displaying a peptide from the N-terminal part of SlpA (SlpA66-215) bound to
the same targets as S-layered L. brevis cells, i.e. to human epithelial cells representing
human gut (Int 407, Caco-2), urinary bladder (T24) and blood vessels (EA-hy926)
and to the immobilized form of the human extracellular matrix protein fibronectin.
Flagella with no inserts and those displaying the C-terminal part of SlpA (SlpA209-417)
failed to bind, confirming binding specificity and delineating the adhesive property to
the N-terminal part of SlpA. The smallest SlpA fragment binding to Int 407 cells was
81 amino acids in size and comprised amino acids 66-146 in the mature SlpA protein.
Neither bacterial cells nor flagella agglutinated human erythrocytes.
The binding of L. brevis cells to Int 407 cells could not be inhibited either by
chimeric flagella or by antibodies from an antiserum raised against whole SlpA or from
an antiserum raised against the histidine-tagged N-terminal binding peptide his-SlpA66-
215
. To explain the lack of inhibition by antibodies and to the verify the location of the
SlpA inserts in the chimeric flagella, the immunological reactivities of the chimeric
flagella, of the N-terminal peptide his-SlpA66-215 and of whole L. brevis cells with the
two antisera were tested using Western hybridization, immunoelectron microscopy or
immunofluorescence microscopy (Figures 1 and 2 in I). These experiments led to two
major conclusions. First, as the anti-peptide antiserum failed to recognize its antigen
on whole L. brevis cells in an immunofluorescence assay, the region encompassing
residues 66-215 of SlpA was demonstrated to be poorly accessible to antibodies in the
polymerized S-layer. Second, the reactivities of the two antisera with the N-terminal
part of SlpA were clearly different. The anti-peptide serum was more reactive, as it
recognized its antigen both from whole SlpA and the chimeric flagella in Western
hybridization and from the flagellar inserts in immunelectron microscopy, while the
anti-SlpA serum recognized only large N-terminal inserts in the flagella in Western
32
hybridization and immunoelectron microscopy. This indicates that the N-terminal

region of SlpA is poorly immunogenic when whole SlpA is used as an immunogen.
In light of these results, the lack of inhibition by antibodies could be explained by
the poor accessibility of the binding region to antibodies from the serums and by the
low amount of binding-site-specific antibodies in the anti-SlpA serum. The lack of
inhibition by chimeric flagella, in turn, could at least partly be explained by the relatively
low concentration of flagella used in the inhibition experiments compared with the
very efficient adherence of bacterial cells having the binding epitope as hundreds
of thousands of copies on their surface. The intriguing immunological findings of
the location of the binding region in SlpA in a rather non-accessible environment
lead at this stage of investigation to the hypothesis of the binding site being located
in a groove or “pocket” in the three-dimensional structure of SlpA not accessible to
antibodies but still able to bind to the receptor on target cells and fibronectin. Further
research has shed more light on the structure and structure-function relationships of
different regions of SlpA (see Sections 4.1.2-4.1.4).
The binding of SlpA to both epithelial cells and fibronectin raised the question
about the role of cell surface fibronectin as a receptor for SlpA. Fibronectin is a large
glycoprotein present in plasma and extracellular fluids, in the extracellular matrix
and on eukaryotic cells, where it specifically binds to a variety of integrin molecules
(Pankov & Yamada, 2002). It has been demonstrated also in the intestine of humans
(Korhonen et al., 2000; Groos et al., 2003) and rodents (Quaroni et al., 1978; Laurie
et al., 1982; Kolachala et al., 2007) and it is produced by cultured human (Nickerson
et al., 2001; Walia et al., 2004; Kolachala et al., 2007) and rodent intestinal epithelial
cell lines (Quaroni et al., 1978; Göke et al., 1996). A number of bacterial species
have fibronectin binding proteins on their surface mediating attachment or invasion
to eukaryotic cells either directly or through a bridging mechanism (Joh et al., 1999;
Monteville et al., 2003; Schwarz-Linek et al., 2004; 2006; Graham et al., 2008).
Fibronectin binding seems to be rather common also in lactobacilli (Toba et al., 1995;
Turner et al., 1999; Kapczynski et al., 2000; Sillanpää, 2001; Styriak et al., 2001;
Lorca et al., 2002; Styriak et al., 2003; Jakava-Viljanen & Palva, 2007; Munoz-
Provencio et al., 2009) but, with the exception of the surface localized enolase of
L. plantarum (Castaldo et al., 2009) (see section 1.2.7), the binding molecules have
usually not been characterized. Only one Lactobacillus protein with a predicted
procaryotic fibronectin-binding domain and a role in bacterial adherence to Caco-2
cells has been identified (Buck et al., 2005). As the synthesis of fibronectin in Caco-2
cells decreases during differentiation (Levy et al., 1994), and in the study of Buck et al.
(2005) the binding of fibronectin of the Lactobacillus strain was not verified, the role
of fibronectin in the Caco-2 cell binding of this strain remains obscure. Kapczynski
et al. (2000) demonstrated that the adherence of Lactobacillus acidophilus to Int 407
cells correlates with the spatial distribution of fibronectin on the surface of the cells.
In the study of Kapczynski et al (2000), the participating Lactobacillus molecule was,
based on inhibition studies, suggested to be a lipoteichoic acid. The binding of L.
brevis through SlpA to epithelial cells might also be partially mediated by fibronectin.
An interaction between SlpA and fibronectin was demonstrated by de Leeuw et al.
(2006) by surface plasmon resonance studies, although soluble fibronectin with a
33
conformation different from that of the immobilized form was used. Fibronectin has
4-9% carbohydrate (Pankov & Yamada, 2002), and the carbohydrate chains might
in principle extend to the shielded N-terminal region of SlpA on bacterial cells.
Furthermore, indications of carbohydrate binding by an S-layered L. brevis strain and
its guanidine hydrochloride extractable surface material have been presented (Uchida
et al., 2006), although aggregates of the S-layer protein were present in the binding
assay compromising its specificity. However, Int 407 cells cultivated as monolayers
express only small amounts of fibronectin on their surfaces (Nickerson et al., 2001),
and the binding of the flagella to fibronectin was very weak (Fig. 4 in I), while the
binding of the chimeric flagella to epithelial cells was efficient. Such sparse and weak
interactions are therefore not likely, at least alone, to be responsible for the observed
very efficient binding of L. brevis to epithelial cells (Table 1 in I). The principal
receptor for SlpA in epithelial cells thus remains to be investigated.
The binding of L. brevis to Caco-2 cells was only moderately inhibited by
the chemical removal of SlpA from L. brevis cells (Table 1 in I), indicating that in
addition to SlpA, L. brevis ATCC 8287 probably has other adhesive surface molecules
contributing to adherence. The expression of multiple adhesins is common in bacterial
pathogens (Holden & Gally, 2004; Wright & Hultgren, 2006; Sillanpää et al., 2008;
Flanagan et al., 2009; Jakubovics et al., 2009; Nicholson et al., 2009) and is likely
to occur in commensal bacteria as well. The contribution of other adhesive factors in
L. brevis adherence to the other cell lines can not be completely excluded either, as
the extraction by guanidium hydrochloride also removes some other, minor proteins
from the cell surface (Fig. 2 in I), and the extraction or loss of SlpA may alter the
conformation and/or functionality of the remaining protein or non-protein components
of the cell wall.
The molecular mechanism of the binding of SlpA to fibronectin or epithelial cells
is currently not known. The demonstration of the binding by flagellar display indicates
that the binding is not critically dependent on the presence of a two-dimensional,
regular S-layer structure, which is not formed in the flagella. As later shown in Study
II, most of the fragments conferring binding in the flagella were unable to form regular
self-assembly products as recombinant proteins. The lack of necessity of the S-layer
structure in adherence has also been confirmed in studies where the N-terminal part
of SlpA, including the adhesive region (SlpA1-217), displayed on the surface of a
non-adhesive Lactococcus strain rendered the strain adhesive to Int 407 cells and
fibronectin (Åvall-Jääskeläinen et al., 2003). Similarly, the collagen-binding property
of the S-layer protein CbsA of Lactobacillus crispatus was preserved when the protein
was displayed on the surface of a non-S-layered, non-adhesive L. casei strain, where
it does not form a regular lattice structure (Martinez et al., 2000). Surface plasmon
resonance experiments have shown that SlpA binds soluble fibronectin with a moderate
affinity (Kd, 89.8 nM) and that the binding is inhibited by a serine protease inhibitor,
benzamidin, while the binding of laminin by SlpA is threefold more efficient and
not inhibited by benzamidin, suggesting different mechanisms and possibly different
regions of SlpA involved (de Leeuw et al., 2006). In the study of de Leeuw et al.
(2006), the hydrophobicity and, specifically, the arginines in the N-terminal region
of SlpA were suggested to contribute to the inhibition of fibronectin binding by the
34
polar benzamidine molecule. Not excluding the potential participation of arginine

molecules in specific interactions, the overall hydrophobicity of SlpA is higher in the
C-terminal than in the N-terminal region (I).
4.1.2 Cell wall binding (II)

Several pieces of evidence suggested the existence of a two-domain organization in
SlpA with an N-terminal cell-wall binding region. Trypsin degradation of SlpA and
the N-terminal sequencing of the proteolytic fragments revealed a protease-resistant
C-terminal part of 246 amino acids and a protease-susceptible N-terminal part in SlpA
(Fig. 3 in II). The same pattern of proteolytic fragments, but at a very low intensity,
was seen after the trypsin degradation of S-layered L. brevis cells. A comparison of
the amino acid sequences of SlpA and five other S-layer proteins of L. brevis strains
revealed that the N-terminal parts were conserved and had a high predicted pI value
(Fig. 1 in II); also the (C-terminal) cell wall binding domains in the S-layer proteins
of L. acidophilus ATCC 4356 and L. crispatus JCM5810 are conserved and highly
positively charged (Smit et al., 2001; Antikainen et al., 2002). Finally, the N-terminal
cell wall binding region in SlpA was confirmed by testing the cell wall binding
capabilities of C- and N-terminally truncated recombinant SlpA proteins, and the first
145 amino acids of mature SlpA alone were found to be sufficient for binding to
isolated cell wall fragments of L. brevis ATCC 8287, while N-terminally truncated
proteins were unable to bind (Fig. 6 in II).
Conserved carbohydrate binding motifs were identified in the positively charged
N-terminal regions of six L. brevis S-layer proteins (Fig. 7 in II). These regions share
similarity with the repeated C-terminal carbohydrate binding sequences detected in
clostridial toxins, streptococcal glucosyltransferases (Wren, 1991; von Eichel-Streiber
et al., 1992) and the S-layer proteins of L. acidophilus group organisms (Smit et al.,
2001) (see also Section 1.2.3). These motifs are supposed to play a general role in
protein-carbohydrate interactions by acting as initial attachment sites between the
protein and the carbohydrate, enabling the specific interactions to occur (von Eichel-
Streiber et al., 1992). They may thus have a role in the observed binding of SlpA to
the glycosylated extracellular matrix protein fibronectin or carbohydrate-containing
epithelial cell receptors (see Section 4.1.1). More likely, however, they contribute
to the anchoring of SlpA to the cell wall polysaccharides. While the S-layer proteins
of L. acidophilus ATCC 4356 and L. crispatus JCM5810 bind to teichoic acids, and
electrostatic interactions were suggested in the binding (Smit et al., 2001; Antikainen
et al., 2002; Smit & Pouwels, 2002), the cell wall receptor of SlpA was in this study
shown to be one other than teichoic or lipoteichoic acid: a treatment removing teichoic
acids had no effect on the ability of the cell walls to bind SlpA, whereas a treatment
affecting wall polysaccharides had, and lipoteichoic acids were not present in the cell
wall preparations used in the binding experiments. The results of this study are thus
in accordance with early studies, in which neutral polysaccharides in the cell walls
of L. brevis ATCC 8287 were suggested to be involved in the anchoring of the SlpA
to the cell wall (Shimohashi et al., 1976; Masuda & Kawata, 1980), but the detailed
structure of this polysaccharide remains to be determined. The location of the cell
wall binding domain in the N-terminal part of SlpA and the dissimilarity of the cell
35
wall receptor apparently reflect the phylogenetic non-relatedness of L. brevis and its
S-layer protein to L. acidophilus group organisms and their S-layer proteins.
4.1.3 Self-assembly (II)

The protease resistance of the C-terminal part of SlpA, the variable amino acid
sequences of the known L. brevis S-layer proteins in this distinct region and the
localization of the cell wall binding domain in the N-terminal part (see Section 4.1.2)
suggested an external location and a role in self-assembly for the C-terminal part of
SlpA. The location of the self-assembly region was confirmed by transmission electron
microscopy of the self-assembly products of N-terminally truncated recombinant
SlpA proteins. In these studies, the protein encompassing amino acids 179-435 in
mature SlpA was able to form a lattice identical to that formed by wild-type SlpA,
as recognized by electron microscopy (Fig. 4 in II) and by the determination of
lattice constants. The removal of 11 residues more from the N-terminus in rSlpA190-435
prevented lattice formation. Surprisingly, also larger N-terminally truncated proteins
rSlpA167-435 and rSlpA149-435 were unable to form regular lattices, indicating that the
extension of the peptide further to the cationic N-terminal region interferes with lattice
formation when the rest of the protein and the cell wall are not present, probably by
steric hindrance and/or by preventing the acquisition of the native conformation of
the peptide.
In conclusion, SlpA was found to be a two-domain protein in which the N-terminal
domain (residues 1-178), including the cell wall binding region, is shielded from the
external environment by the protease-resistant C-terminal domain (residues 179-
435), responsible for the self-assembly of the SlpA monomers to a regular layer. SlpA
thus follows the current view of Gram-positive S-layer proteins having two separate
functional domains. However, the order of the functional regions is the opposite of the
other thus far characterized Lactobacillus S-layer proteins. A schematic presentation
of the structure of SlpA is shown in Fig. 4.
Cell wall
binding (1-178) Self-assembly (179-435)
1 435
Fn (66-215) and
epithelial cell (66-146)
binding
Figure 4. A schematic presentation of the functional regions in mature SlpA. The different
colours of the ten amino acid stretches shown by boxes indicate the level of similarity between
six L. brevis S-layer proteins: white, 0-20%; dark grey, 61-80% identical or similar amino acids
in a stretch. Short black bars indicate the regions most accessible in the assembled S-layer, as
detected by maleimide modification (III). Adapted from Figure 1 of (II) with permission of the
publisher.
36
4.1.4 Locations of individual residues (III)

Considering potential applications of SlpA as a display vehicle of foreign peptides, a
detailed mapping of the locations of individual residues in the self-assembly region
of SlpA was performed by cysteine scanning mutagenesis and targeted chemical
modification. A series of 46 SlpA mutant proteins was generated with 40 of the single,
novel cysteine residues located in the C-terminal self-assembly region. The abilities
of the mutant proteins to self-assemble into regular lattices in solution and on cell
wall fragments was confirmed by transmission electron microscopy and by SAXS
combined with cryoelectron microscopy, respectively (Figures 1 and 2 in III). The
surface accessibility of the mutated residues was studied using two sulfhydryl-reactive
maleimide reagents of different molecular weights, TMM(PEG)12 and AlexaFluor488-
maleimide. Their reactions with both monomeric proteins in solution and with proteins
assembled on cell wall fragments were investigated.
Using the combined results of the different experimental settings (Figures 4
and 5 in III) and previous data about the domain organization of SlpA (Study II),
the mutated residues were assigned to four groups according to their most probable
location in the protein monomer and the lattice structure: those on the outer surface
of the lattice, those in the protein interior, those on the inner surface of the lattice and
those on the subunit interface/pore region of the lattice (Table 2 in III). The latter two
groups were characterized by a different extent of modification of the cysteine residue
in the monomeric and assembled form, and were differentiated from each other on the
basis of the location of the residues, the “inner surface” group consisting of residues
of the cell wall binding region and the extreme N-terminal part of the self-assembly
region. The assignments to the four groups were supported by several facts. The
groups were clearly distinguishable by the characteristic amino acid compositions of
the regions surrounding the mutated residues: residues mapped to the outer surface
were in a more hydrophilic and negatively charged environment than those mapped
to the protein interior; the surroundings of the residues mapped to the inner surface
were positively charged, and the environments of the interface/pore group residues
were distinguishable by the lack of charge (Table 2 in III). The assignments were
also supported by the sites of predicted, but not observed, trypsin cleavage, most
of which were located at sites predicted to be in the protein interior or pore region
(Fig. 6 in III). In addition, the assignments were in agreement with the PredictProtein
secondary structure prediction of SlpA (Rost et al., 2004), chosen on the basis that it
most accurately reproduces the secondary structure estimates obtained by physical
measurements (Mobili et al., 2009b): residues assigned to the outer surface of the
lattice were mostly located in the predicted loops, residues assigned to protein interior
within the predicted β-strands and residues assigned to the pore/interface regions
within both loops and β-strands (Fig. 6 in III).
The mutated residues that were most accessible in the assembled S-layer were
located in four segments of SlpA spanning amino acids 256-273, 303-316, 335-
349 and 362-372 (Figures 4 and 5 in III). This finding is in good accordance with a
previous study, in which an 11-amino-acid poliovirus VP1 epitope was displayed in
SlpA (Åvall-Jääskeläinen et al., 2002): of the three insertion sites in the self-assembly
region of SlpA tested, the epitope was accessible to antibodies when inserted between
37
residues 251 and 252 (near the first surface-accessible segment of Study III) or between
residues 365 and 366 (within the last surface-accessible segment of Study III). The
inability of antibodies to recognize the epitope inserted between residues 316 and 317,
which is an accessible site as measured by maleimide modification in Study III, may
be due to the altered conformation of the inserted peptide or SlpA at the insertion site,
or to the possibly different interactions in antibody binding compared with those in a
cysteine-maleimide reaction.
Overall, the results of the cysteine scanning mutagenesis study confirm the two-
domain organization of SlpA. The residues mapped to the outer surface of the layer
may have potential in further applications, e.g. in the surface display of antigenic or
other effector molecules in SlpA. Furthermore, as the amino acid sequences of S-layer
proteins usually fit poorly the existing algorithms for the prediction of secondary
structures or solvent accessibilities, the relationships found between the primary
structure and surface accessibilities of residues in SlpA could prove helpful when
developing a program for reliably predicting S-layer protein structures.
4.2. Activities of slpA promoters (IV)
The gene encoding SlpA is preceded by two consecutive promoters, P1 and P2

(Vidgren et al., 1992). Previous studies have indicated that both of these promoters are
functional during all growth phases in L. brevis ATCC 8287, with significantly more
transcripts detected from the downstream promoter P2, and the expression of slpA has
been suggested to be tightly regulated (Kahala et al., 1997). As Lactobacillus S-layer
proteins are attractive candidates for display vehicles of antigens or other effector
molecules in humans or animals and the factors affecting the expression of S-layer
protein genes in lactobacilli are poorly known, a study aiming at the characterization
of the function of the two promoters in more detail and at the recognition of potential
cis-acting elements upstream of L. brevis slpA was initiated. The strategy was to
separate the two promoters from each other on reporter plasmids with different
parts of their upstream sequences deleted (Fig. 1 in IV), and to measure the enzyme
activities conferred by the different promoter regions (Fig. 2 in IV). Moreover,
promoter activities were investigated at the mRNA level (Fig. 3 in IV), effects of
selected intestinal conditions on promoter activities were evaluated in vitro (Fig. 4 in
IV) and sequence analyses of the upstream regions of slpA and other Lactobacillus
S-layer protein genes were performed.
As shown in Fig. 2 in Study IV, in L. lactis, only promoter 1 was recognized,
and the reporter enzyme activities of the L. lactis strains carrying the smallest region
upstream of P1, 57 base pairs above the transcription start, were significantly lower
than the activities of strains carrying larger promoter regions. In L. brevis, both
promoters were recognized during all growth phases, as expected, and the wild-type
double promoter was more efficient than either P1 or P2 alone. However, no difference
was observed between the efficiencies of the two promoters, except that the reporter
enzyme activity of the strain carrying the smallest region upstream of P1 was somewhat
lower than the very high activities of all of the other strains. This conflicting result
compared with a previous study (Kahala et al., 1997) is probably attributed to the
different methods used in these two studies. In the study of Kahala et al. (1997), the
38
slpA mRNAs were quantified from L. brevis by a method based on the hybridization
of two oligonucleotide probes specific for either P1 transcript or both transcripts; this
method is prone to errors due to possible differences in the binding efficiencies of
the two probes and the relatively poor linearity of the immunological digoxigenin-
based detection (unpublished observation). In the present work, two methods, the
highly reproducible TRAC (transcript analysis with aid of affinity capture) (Rautio
et al., 2008) and conventional Northern blotting, were used to quantify reporter gene
transcripts from L. brevis clones carrying different slpA promoter regions on plasmids.
Essentially the same pattern of promoter activities seen in the L. brevis clones at a
reporter enzyme level was also seen at the mRNA level, suggesting regulation of slpA
expression at the transcriptional rather than at the translational level (Fig. 3 in IV).
The high activities of the promoters were retained when the recombinant L. brevis
strains were cultivated under conditions mimicking the intestinal environment (Fig. 4
in IV). No indications of an effect of the composition of the growth medium on slpA
gene expression in L. brevis were obtained.
Three of the potential regulatory motifs, originally identified by Wels et al.
(2006) in the upstream regions of conserved genes in bacilli and lactic acid bacteria,
were also detected in the upstream, intergenic region of slpA (Fig. 1B in IV). None
of the three motifs was, however, located in the region missing from the smallest
P1 promoter construct, and the motifs could thus not directly explain the observed
patterns of promoter activities. However, a few pieces of evidence suggest that cis-
acting factors may be involved in the regulation of slpA expression in L. brevis ATCC
8287 under some, as yet unidentified conditions. All of the three potential regulatory
motifs identified were found also in the genome of L. brevis ATCC 14869 upstream
of the S-layer protein gene slpB, but not upstream of slpC or slpD of the same strain;
slpB and slpD are expressed under different conditions, the differential expression
probably being mediated by a soluble cytoplasmic factor, and slpC is known to be
silent under laboratory conditions (Jakava-Viljanen et al., 2002). The upstream region
of slpA is well conserved in a subset of L. brevis strains, as the sequences upstream
of slpA, slpB and the truncated slpA homologue in L. brevis ATCC 367 are practically
identical, although the similarity between the coding regions of slpA and slpB is only
30% (Jakava-Viljanen et al., 2002). Finally, upstream of slpA, an ORF with similarity
to bacterial genes encoding aminotransferases with helix-turn-helix DNA-binding
domains (so-called MocR-type GntR-family transcriptional regulators) has been
identified (unpublished data), though the possible participation of this gene in the
regulation of slpA expression is currently highly hypothetical.
In summary, the present information about the function of the two promoters of
slpA indicates a very efficient function of both promoters during all growth phases
in L. brevis, but not in Lactococcus lactis. More upstream sequence is needed for the
full activity of promoter 1 than for promoter 2 in L. brevis. The possible regulation
of slpA expression occurs at the transcriptional level, and cis-acting factors are likely
to participate. The two promoters retain their activities under experimental intestinal
conditions in vitro, which is advantageous for the potential use of SlpA as a carrier of
foreign molecules in applications in which bacterial replication in the gastrointestinal
tract is desired, as in live oral vaccines.
39
Conclusions
5. CONCLUSIONS
In this work, the structure and function of the S-layer protein SlpA of Lactobacillus
brevis ATCC 8287 and the use of the promoters of its gene were studied. S-layer
proteins have a wide application potential in nanobiotechnology. As food-grade and
potentially probiotic organisms, lactobacilli are also excellent candidates for health-
related applications, where their abilities to survive in the gastrointestinal tract could
be utilized in the design of live oral vaccines, and their S-layer proteins could be
used as carriers of antigens or other medically important molecules. A comprehensive
study of bacterial S-layers and their expression is thus warranted.
This work supported the view of Gram-positive S-layer proteins as two-domain
entities, where one domain is responsible for cell wall binding, and the other for the
self-assembly of the regular surface layer. The common theme of carbohydrates as the
binding sites for S-layer proteins in the cell walls of Gram-positive bacteria was also
supported. In the S-layer protein SlpAof Lactobacillus brevisATCC 8287, the N-terminal
domain was found to bind to the cell wall and the C-terminal domain to be responsible
for self-assembly; the domains were thus in a reversed order compared with the other
thus far characterized Lactobacillus S-layer proteins, those of two phylogenetically
distant, L. acidophilus-related species (Smit et al., 2001; Antikainen et al., 2002). Also
the binding mechanism of SlpA to the cell wall independently of teichoic acids was
found to be unique among the Lactobacillus S-layer proteins examined to date. The
structures of the polysaccharides in the cell wall of L. brevis ATCC 8287 are currently
being investigated. As at the moment only three Lactobacillus S-layer proteins have
been structurally and functionally thoroughly studied, different types of structure-
function relationships and cell wall binding mechanisms will presumably be revealed
as more Lactobacillus S-layer proteins are characterized. Biophysical methods are
increasingly utilized in the structural studies of S-layers, and together with computer
modelling-based methods will probably allow for more high-resolution structures of
bacterial S-layer proteins, which currently are scarce owing to difficulties in obtaining
high-quality crystals for X-ray crystallography. To investigate the S-layer formed by
SlpA in detail, comparative SAXS studies of the SlpA lattice on bacterial cells and of
SlpA reassembled in solution and on different biological surfaces have already been
performed (P. Jääskeläinen, personal communication). Also the solution structure of
the C-terminal part of SlpA has been determined by SAXS (Serimaa et al., 2009), and
attempts to crystallize parts of SlpA are under way. SlpA of L. brevis ATCC 8287 is
currently the only structurally and functionally characterized S-layer protein with a
demonstrated fibronectin-binding function. Further studies are needed to determine
the biological importance of the epithelial cell and fibronectin binding functions of
SlpA, and the elucidation of the structure of SlpA at high resolution would be helpful
in understanding the intriguing location of the binding site in the shielded N-terminal
domain.
With regard to applications, several findings of this study are important. Surface-
exposed regions in the assembled form of SlpA were determined. Surface display
utilizing an S-layer protein as a carrier results in the simultaneous expression of the
foreign peptide as hundreds of thousands of regularly arranged copies on the living
40
Conclusions
cell. Since a clear relationship exists between antigen expression levels and immune
response (Grangette et al., 2001; Seegers, 2002), and Lactobacillus cells as well as
surface layer arrays have intrinsic adjuvant properties (Smith et al., 1993; Miettinen
et al., 1996; Maassen et al., 2000; Seegers, 2002), the identified surface-located
residues are especially attractive candidates for the insertion of foreign epitopes for
live vaccine design. In the future, the simultaneous display of immunomodulating
molecules in the S-layer could be used to further enhance or direct the immune
response. The final suitability of the sites for each application, however, remains to be
experimentally determined; studies aiming at the display of an F18 fimbrial adhesin
fragment in SlpA are currently in progress. The preservation of high activities of the
slpA promoters under conditions mimicking the intestinal environment is essential
in live oral vaccine applications, where bacterial replication in vivo is desired. The
oral delivery route of vaccines is simple and safe and efficiently induces mucosal
immunity, which is ideal for preventing the initial infection of most pathogens (Ryan
et al., 2001; Seegers, 2002; Wells & Mercenier, 2008). Furthermore, as antigen carrier
systems can be significantly improved by the co-display of adhesins (Cano et al.,
1999; Liljeqvist et al., 1999), the epithelial cell and fibronectin binding function of
the N-terminal part of SlpA could be utilized in the targeted delivery of antigenic
or other effector molecules. Preliminary experiments have been performed utilizing
SlpA as a purified immunoglobulin binding fusion protein to target antibodies to the
intestinal surfaces of calves to prevent neonatal diarrhoea by passive immunization
(Khang et al., 2009). Fusion partners in recombinant SlpA proteins produced in E.
coli might further include various therapeutics, such as immunoglobulins, enzymes,
bacteriocides or anti-adhesive agents, which could prove effective on different mucosal
surfaces. Considering other biotechnological applications, the identified surface-
located residues could be modified for the immobilization of bacterial cells or the
S-layer protein, and the finding that residues on the inner surface of the SlpA lattice
are positively charged suggests that immobilization of SlpA on a negatively charged
support without further modification is possible. Immobilization of bacterial cells or
S-layers combined with the display of foreign molecules in the S-layer forms the basis
for the development of different solid-phase reagents, such as biocatalysts, diagnostic
devices, biosensors and biosorbents; in these applications also the thermostability of
SlpA (Mobili et al., 2009b) may be advantageous. The results of this study thus form a
basis for the development of SlpA to become a tool for a wide range of biotechnological
applications.
41
Acknowledgements
6. ACKNOWLEDGEMENTS
This study was carried out at the Faculties of Biosciences and Veterinary Medicine,
University of Helsinki, and supported by the University of Helsinki, the Academy of
Finland, the Ministry of Agriculture and Forestry and the European Union. I am grateful
to Professors Timo Korhonen, Kielo Haahtela and Airi Palva for the opportunity to
perform this work, to Professors Timo Korhonen and Airi Palva and especially Dr.
Ilkka Palva for supervision, and to the reviewers Professor Neil Fairweather and
Professor Per Saris for critically reading the manuscript. I also thank all co-authors,
previous and present collaborators, especially Airi’s and Timo’s S-layer groups, and
other staff at both faculties for co-operation and valuable advice. A special thanks goes
to Dr Jouko Sillanpää for continuous professional support and friendship. Finally, I’m
warmly grateful to my family and relatives for patience, encouragement and support.
Helsinki 2009
42
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Structural and Functional Characterization of The Surface Layer Protein of Lactobacillus Brevis ATCC 8287

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Structural and Functional Characterization of The Surface Layer Protein of Lactobacillus Brevis ATCC 8287

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Structural and functional characterization of the surface layer protein of Lactobacillus brevis ATCC 8287

Structural and functional characterization

Department of Basic Veterinary Sciences

Structural and functional characterization

To be presented, with the permission of the Faculty of Veterinary Medicine,

Professor Airi Palva

Reviewers Professor Per Saris

Professor Neil F. Fairweather

Opponent Professor Atte von Wright

Layout: Tinde Päivärinta

ISBN 978-952-92-6380-6 (paperback)

I Hynönen, U., B. Westerlund-Wikström, A. Palva, and T. K. Korhonen.

II Åvall-Jääskeläinen, S., U. Hynönen, N. Ilk, D. Pum, U. B. Sleytr, and A.

III Vilen, H., U. Hynönen, H. Badelt-Lichtblau, N. Ilk, P. Jääskeläinen, M.

IV Hynönen, U., S. Åvall-Jääskeläinen, and A. Palva. 2009. Characterization

1. REVIEW OF THE LITERATURE

1.1.1 Structure of the Gram-positive cell wall

1.1.2 Surface layer proteins

1.1.2.1 Occurrence, general features and study methods

“signature” for identifying a protein as an S-layer protein is lacking, the unambiguous

1.1.2.2 Expression of S-layer protein genes

1.1.2.3 Cell wall binding and self-assembly regions

of S-layer proteins of Gram-positive bacteria are summarized in Table 2.

Table 2. Self-assembly regions in S-layer proteins of Gram-positive bacteria.

Strain S-layer Location of crystallization Reference

genetically fused to e.g. enzymes, streptavidin, specific antibody fragments, green

1.2. Lactobacilli and their S-layer proteins

Lactic acid bacteria are Gram-positive, non-pathogenic micro-organisms characterized

1.2.1 Occurrence and general properties of Lactobacillus S-layer proteins

1.2.2 Expression of Lactobacillus S-layer protein genes

containing carbohydrate-binding motifs of clostridial toxins and streptococcal

the self-assembly products of CbsA fragments are seen accompanying a mutation of

1.2.4 Functions of Lactobacillus S-layer proteins

directly demonstrated. The haemagglutinating activity of L. acidophilus JCM 1034

1.2.5. Applications of Lactobacillus S-layer proteins

Another potential application is the use of S-layers or S-layered lactobacilli as

1.2.6.1. Cysteine scanning mutagenesis and sulfhydryl chemistry

1.2.6.2 Flagellar display

1.2.7 Non-S-layer adhesive surface proteins of lactobacilli

Table 3. Genetically and functionally characterized non-S-layer adhesive surface

pilus assembly process, a pilin-specific sortase is involved (Ton-That & Schneewind,

2. AIMS OF THE STUDY

As food-grade organisms able to survive, persist and potentially exert health-promoting

Specific aims were as follows:

3. MATERIALS AND METHODS

Strain, plasmid or cell Origin / relevant Study Reference or source

Table 5. Methods used in Studies I-IV

Method Used and

4. RESULTS AND DISCUSSION

4.1. Structure and function of SlpA

4.1.1 Adhesive functions (I)

hybridization and immunoelectron microscopy. This indicates that the N-terminal

polar benzamidine molecule. Not excluding the potential participation of arginine

4.1.2 Cell wall binding (II)

4.1.3 Self-assembly (II)

4.1.4 Locations of individual residues (III)

4.2. Activities of slpA promoters (IV)

The gene encoding SlpA is preceded by two consecutive promoters, P1 and P2

Boekhorst J, Wels M, Kleerebezem M & Siezen RJ (2006b) The predicted secretome of

Cuadros C, Lopez-Hernandez FJ, Dominguez AL, McClelland M & Lustgarten J (2004)

Granato D, Bergonzelli GE, Pridmore RD, Marvin L, Rouvet M & Corthesy-Theulaz IE

Konstantinov SR, Smidt H, de Vos WM et al. (2008) S layer protein A of Lactobacillus

Lupas A, Engelhardt H, Peters J, Santarius U, Volker S & Baumeister W (1994) Domain